Sample records for coronavirus envelope protein

  1. Envelope Protein Palmitoylations Are Crucial for Murine Coronavirus Assembly▿

    PubMed Central

    Boscarino, Joseph A.; Logan, Hillary L.; Lacny, Jason J.; Gallagher, Thomas M.

    2008-01-01

    The coronavirus assembly process encloses a ribonucleoprotein genome into vesicles containing the lipid-embedded proteins S (spike), E (envelope), and M (membrane). This process depends on interactions with membranes that may involve palmitoylation, a common posttranslational lipidation of cysteine residues. To determine whether specific palmitoylations influence coronavirus assembly, we introduced plasmid DNAs encoding mouse hepatitis coronavirus (MHV) S, E, M, and N (nucleocapsid) into 293T cells and found that virus-like particles (VLPs) were robustly assembled and secreted into culture medium. Palmitate adducts predicted on cysteines 40, 44, and 47 of the 83-residue E protein were then evaluated by constructing mutant cDNAs with alanine or glycine codon substitutions at one or more of these positions. Triple-substituted proteins (E.Ts) lacked palmitate adducts. Both native E and E.T proteins localized at identical perinuclear locations, and both copurified with M proteins, but E.T was entirely incompetent for VLP production. In the presence of the E.T proteins, the M protein subunits accumulated into detergent-insoluble complexes that failed to secrete from cells, while native E proteins mobilized M into detergent-soluble secreted forms. Many of these observations were corroborated in the context of natural MHV infections, with native E, but not E.T, complementing debilitated recombinant MHVs lacking E. Our findings suggest that palmitoylations are essential for E to act as a vesicle morphogenetic protein and further argue that palmitoylated E proteins operate by allowing the primary coronavirus assembly subunits to assume configurations that can mobilize into secreted lipid vesicles and virions. PMID:18184706

  2. Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

    PubMed Central

    Kuo, Lili; Hurst-Hess, Kelley R.; Koetzner, Cheri A.

    2016-01-01

    ABSTRACT The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its

  3. [Cell entry mechanisms of coronaviruses].

    PubMed

    Taguchi, Fumihiro; Matsuyama, Shutoku

    2009-12-01

    Enveloped viruses enter into cells via fusion of their envelope and cellular membrane. Spike (S) protein of coronavirus (CoV) is responsible for entry events. We studied the cell entry mechanisms of two different CoVs, murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV). MHV-JHM that induces syncytia in infected cells entered directly from cell surface, i.e., fusion of envelope and plasma membrane, whereas SARS-CoV and MHV-2 that fail to induce syncytia entered via endosome in a protease-dependent fashion, i.e., fusion of envelope and endosomal membrane. The latter viruses entered directly from cell surface, when receptor-bound viruses were treated with proteases that activate fusion activity of their S proteins. The entry pathway of SARS-CoV could influence the severity of the disease. It was also reveled that a highly neurovirulent JHM spread in a receptor-independent fashion, which could result in a high neuropathogenicity of the virus.

  4. Importance of Conserved Cysteine Residues in the Coronavirus Envelope Protein▿

    PubMed Central

    Lopez, Lisa A.; Riffle, Ambere J.; Pike, Steven L.; Gardner, Douglas; Hogue, Brenda G.

    2008-01-01

    Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production. PMID:18184703

  5. Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

    PubMed Central

    Torres, Jaume; Maheswari, Uma; Parthasarathy, Krupakar; Ng, Lifang; Liu, Ding Xiang; Gong, Xiandi

    2007-01-01

    The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM α-helical bundle. We have shown previously that the topology of the α-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM α-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric α-helical bundle model. PMID:17766393

  6. Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

    PubMed Central

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  7. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    PubMed Central

    Torres, Jaume; Surya, Wahyu; Li, Yan; Liu, Ding Xiang

    2015-01-01

    Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i) the envelope protein in coronaviruses and (ii) the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity. PMID:26053927

  8. [Nosocomial infections due to human coronaviruses in the newborn].

    PubMed

    Gagneur, A; Legrand, M C; Picard, B; Baron, R; Talbot, P J; de Parscau, L; Sizun, J

    2002-01-01

    Human coronaviruses, with two known serogroups named 229-E and OC-43, are enveloped positive-stranded RNA viruses. The large RNA is surrounded by a nucleoprotein (protein N). The envelop contains 2 or 3 glycoproteins: spike protein (or protein S), matrix protein (or protein M) and a hemagglutinin (or protein HE). Their pathogen role remains unclear because their isolation is difficult. Reliable and rapid methods as immunofluorescence with monoclonal antibodies and reverse transcription-polymerase chain reaction allow new researches on epidemiology. Human coronaviruses can survive for as long as 6 days in suspension and 3 hours after drying on surfaces, suggesting that they could be a source of hospital-acquired infections. Two prospective studies conducted in a neonatal and paediatric intensive care unit demonstrated a significant association of coronavirus-positive nasopharyngal samples with respiratory illness in hospitalised preterm neonates. Positive samples from staff suggested either a patient-to-staff or a staff-to-patient transmission. No cross-infection were observed from community-acquired respiratory-syncitial virus or influenza-infected children to neonates. Universal precautions with hand washing and surface desinfection could be proposed to prevent coronavirus transmission.

  9. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

    2014-01-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  10. The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

    PubMed Central

    McBride, Ruth; Fielding, Burtram C.

    2012-01-01

    A respiratory disease caused by a novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), was first reported in China in late 2002. The subsequent efficient human-to-human transmission of this virus eventually affected more than 30 countries worldwide, resulting in a mortality rate of ~10% of infected individuals. The spread of the virus was ultimately controlled by isolation of infected individuals and there has been no infections reported since April 2004. However, the natural reservoir of the virus was never identified and it is not known if this virus will re-emerge and, therefore, research on this virus continues. The SARS-CoV genome is about 30 kb in length and is predicted to contain 14 functional open reading frames (ORFs). The genome encodes for proteins that are homologous to known coronavirus proteins, such as the replicase proteins (ORFs 1a and 1b) and the four major structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). SARS-CoV also encodes for eight unique proteins, called accessory proteins, with no known homologues. This review will summarize the current knowledge on SARS-CoV accessory proteins and will include: (i) expression and processing; (ii) the effects on cellular processes; and (iii) functional studies. PMID:23202509

  11. The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

    PubMed Central

    Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; DeDiego, Marta L.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Enjuanes, Luis

    2014-01-01

    A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation. PMID:25122212

  12. Purified coronavirus Spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

    PubMed Central

    Mu, Haiyan; Taylor, Justin K; Massare, Michael; Flyer, David C

    2014-01-01

    Development of vaccination strategies for emerging pathogens are particularly challenging because of the sudden nature of the emergence of these viruses and the long process needed for traditional vaccine development. Therefore, there is a need for development of a rapid method of vaccine development that can respond to emerging pathogens in a short time frame. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and Middle East respiratory syndrome (MERS-CoV) in late 2012 demonstrate the importance of coronaviruses as emerging pathogens. The spike glycoproteins of coronaviruses reside on the surface of the virion and are responsible for virus entry. The spike glycoprotein is the major immunodominant antigen of coronaviruses and has proven to be an excellent target for vaccine designs that seek to block coronavirus entry and promote antibody targeting of infected cells. Vaccination strategies for coronaviruses have involved live attenuated virus, recombinant viruses, non-replicative virus-like particles expressing coronavirus proteins or DNA plasmids expressing coronavirus genes. None of these strategies has progressed to an approved human coronavirus vaccine in the ten years since SARS-CoV emerged. Here we describe a novel method for generating MERS-CoV and SARS-CoV full-length spike nanoparticles, which in combination with adjuvants are able to produce high titer antibodies in mice. PMID:24736006

  13. Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein.

    PubMed

    Kuo, Lili; Koetzner, Cheri A; Hurst, Kelley R; Masters, Paul S

    2014-04-01

    The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective

  14. Accessory proteins of SARS-CoV and other coronaviruses.

    PubMed

    Liu, Ding Xiang; Fung, To Sing; Chong, Kelvin Kian-Long; Shukla, Aditi; Hilgenfeld, Rolf

    2014-09-01

    The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)). Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

    PubMed

    Hsin, Wei-Chen; Chang, Chan-Hua; Chang, Chi-You; Peng, Wei-Hao; Chien, Chung-Liang; Chang, Ming-Fu; Chang, Shin C

    2018-05-24

    Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969) ME ), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969) ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969) ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

  16. Genotyping bovine coronaviruses.

    USDA-ARS?s Scientific Manuscript database

    Bovine coronaviruses (BoCV) are enveloped, single-stranded, positive-sense RNA viruses of the Coronaviridae family. Infection is associated with enteritis and pneumonia in calves and Winter Dysentery in adult cattle. Strains, isolated more than 50 years ago, are used in vaccines and as laboratory ...

  17. Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and Its Implication in Virion Assembly and Budding

    PubMed Central

    Wang, Jibin; Fang, Shouguo; Xiao, Han; Chen, Bo; Tam, James P.; Liu, Ding Xiang

    2009-01-01

    Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus. PMID:19287488

  18. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

    PubMed

    Wang, Sheng-Fan; Tseng, Sung-Pin; Yen, Chia-Hung; Yang, Jyh-Yuan; Tsao, Ching-Han; Shen, Chun-Wei; Chen, Kuan-Hsuan; Liu, Fu-Tong; Liu, Wu-Tse; Chen, Yi-Ming Arthur; Huang, Jason C

    2014-08-22

    The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M.

    PubMed

    Oostra, M; de Haan, C A M; de Groot, R J; Rottier, P J M

    2006-03-01

    The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N

  20. The SARS coronavirus nucleocapsid protein--forms and functions.

    PubMed

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    PubMed

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

  2. [Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].

    PubMed

    Lu, Jian; Zhou, Bai-ping; Zhou, Yu-sen; Jiang, Xiao-ling; Wen, Li-xia; Le, Xiao-hua; Li, Bing; Xu, Liu-mei; Li, Li-xiong

    2005-03-01

    To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS. SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly. The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates. The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.

  3. Conserved antigenic sites between MERS-CoV and Bat-coronavirus are revealed through sequence analysis.

    PubMed

    Sharmin, Refat; Islam, Abul B M M K

    2016-01-01

    MERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV. Multiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein's antigenic sites are found to be conserved with those in HKU4 and HKU5. This sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity.

  4. Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

    PubMed

    Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

    2007-03-01

    Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

  5. Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function.

    PubMed

    Jaimes, Javier A; Whittaker, Gary R

    2018-04-01

    Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

    PubMed

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

    2015-09-01

    RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression

  7. CORONAVIRUS VIRULENCE GENES WITH MAIN FOCUS ON SARS-CoV ENVELOPE GENE

    PubMed Central

    DeDiego, Marta L.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis

    2014-01-01

    Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and 7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal

  8. Coronavirus virulence genes with main focus on SARS-CoV envelope gene.

    PubMed

    DeDiego, Marta L; Nieto-Torres, Jose L; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis

    2014-12-19

    Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and -7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal

  9. Severe Acute Respiratory Syndrome Coronaviruses with Mutations in the E Protein Are Attenuated and Promising Vaccine Candidates

    PubMed Central

    Regla-Nava, Jose A.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Fernandez-Delgado, Raul; Fett, Craig; Castaño-Rodríguez, Carlos; Perlman, Stanley; DeDiego, Marta L.

    2015-01-01

    ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates. IMPORTANCE Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses

  10. SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

    PubMed Central

    2014-01-01

    Background Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. Results SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. Conclusions The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly. PMID:24766657

  11. SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.

    PubMed

    Tseng, Ying-Tzu; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

    2014-04-27

    Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.

  12. Mechanism of protein import across the chloroplast envelope.

    PubMed

    Chen, K; Chen, X; Schnell, D J

    2000-01-01

    The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

  13. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

    PubMed

    Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

    2010-05-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

  14. Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy

    PubMed Central

    Walls, Alexandra C.; Tortorici, M. Alejandra; Frenz, Brandon; Snijder, Joost; Li, Wentao; Rey, Félix A.; DiMaio, Frank; Bosch, Berend-Jan; Veesler, David

    2017-01-01

    The threat of a major coronavirus pandemic urges the development of suitable strategies to combat these pathogens. HCoV-NL63 is an α-coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. We report here the 3.4 Å resolution cryo-electron microscopy reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass-spectrometry, provides a structural framework to understand accessibility to antibodies. The structure also reveals a remarkable modular architecture of the receptor-binding subunit and the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on masking of epitopes with glycans and activating conformational changes, to evade the immune system of infected hosts. PMID:27617430

  15. SARS-unique fold in the Rousettus bat coronavirus HKU9.

    PubMed

    Hammond, Robert G; Tan, Xuan; Johnson, Margaret A

    2017-09-01

    The coronavirus nonstructural protein 3 (nsp3) is a multifunctional protein that comprises multiple structural domains. This protein assists viral polyprotein cleavage, host immune interference, and may play other roles in genome replication or transcription. Here, we report the solution NMR structure of a protein from the "SARS-unique region" of the bat coronavirus HKU9. The protein contains a frataxin fold or double-wing motif, which is an α + β fold that is associated with protein/protein interactions, DNA binding, and metal ion binding. High structural similarity to the human severe acute respiratory syndrome (SARS) coronavirus nsp3 is present. A possible functional site that is conserved among some betacoronaviruses has been identified using bioinformatics and biochemical analyses. This structure provides strong experimental support for the recent proposal advanced by us and others that the "SARS-unique" region is not unique to the human SARS virus, but is conserved among several different phylogenetic groups of coronaviruses and provides essential functions. © 2017 The Protein Society.

  16. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

    PubMed Central

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

    2012-01-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

  17. Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins▿

    PubMed Central

    Haynes, Lia M.; Miao, Congrong; Harcourt, Jennifer L.; Montgomery, Joel M.; Le, Mai Quynh; Dryga, Sergey A.; Kamrud, Kurt I.; Rivers, Bryan; Babcock, Gregory J.; Oliver, Jennifer Betts; Comer, James A.; Reynolds, Mary; Uyeki, Timothy M.; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M.; Anderson, Larry J.

    2007-01-01

    Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies. PMID:17229882

  18. Viral metagenomics, protein structure, and reverse genetics: Key strategies for investigating coronaviruses.

    PubMed

    Johnson, Bryan A; Graham, Rachel L; Menachery, Vineet D

    2018-04-01

    Viral metagenomics, modeling of protein structure, and manipulation of viral genetics are key approaches that have laid the foundations of our understanding of coronavirus biology. In this review, we discuss the major advances each method has provided and discuss how future studies should leverage these strategies synergistically to answer novel questions. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

    PubMed Central

    Nakagawa, K.; Lokugamage, K.G.; Makino, S.

    2017-01-01

    Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

  20. Phage-display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential

    USDA-ARS?s Scientific Manuscript database

    The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell recepto...

  1. Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor

    PubMed Central

    Babcock, Gregory J.; Esshaki, Diana J.; Thomas, William D.; Ambrosino, Donna M.

    2004-01-01

    A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S1190). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S1190 binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S1190 maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S1190 glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins. PMID:15078936

  2. Structural model of the SARS coronavirus E channel in LMPG micelles.

    PubMed

    Surya, Wahyu; Li, Yan; Torres, Jaume

    2018-06-01

    Coronaviruses (CoV) cause common colds in humans, but are also responsible for the recent Severe Acute, and Middle East, respiratory syndromes (SARS and MERS, respectively). A promising approach for prevention are live attenuated vaccines (LAVs), some of which target the envelope (E) protein, which is a small membrane protein that forms ion channels. Unfortunately, detailed structural information is still limited for SARS-CoV E, and non-existent for other CoV E proteins. Herein, we report a structural model of a SARS-CoV E construct in LMPG micelles with, for the first time, unequivocal intermolecular NOEs. The model corresponding to the detergent-embedded region is consistent with previously obtained orientational restraints obtained in lipid bilayers and in vivo escape mutants. The C-terminal domain is mostly α-helical, and extramembrane intermolecular NOEs suggest interactions that may affect the TM channel conformation. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. About Coronavirus

    MedlinePlus

    ... Coronaviruses Symptoms and Diagnosis Transmission Prevention and Treatment Human Coronavirus Types SARS-CoV MERS-CoV Resources and References About Coronaviruses Recommend on Facebook Tweet Share Compartir Symptoms and Diagnosis Lists illnesses ...

  4. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusivemore » protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.« less

  5. Expression, post-translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus.

    PubMed

    Le, Tra M; Wong, Hui H; Tay, Felicia P L; Fang, Shouguo; Keng, Choong-Tat; Tan, Yee J; Liu, Ding X

    2007-08-01

    The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site-directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N-linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b-induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

  6. DOE Office of Scientific and Technical Information (OSTI.GOV)

    McBride, Corrin E., E-mail: cmcbrid5@jhmi.ed; Machamer, Carolyn E., E-mail: machamer@jhmi.ed

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation ofmore » S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.« less

  7. Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells.

    PubMed

    Tooze, J; Tooze, S A; Fuller, S D

    1987-09-01

    Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.

  8. A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

    PubMed

    Russell, R L; Rohrmann, G F

    1990-01-01

    A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

  9. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution

    PubMed Central

    Zeng, Qinghong; Langereis, Martijn A.; van Vliet, Arno L. W.; Huizinga, Eric G.; de Groot, Raoul J.

    2008-01-01

    The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer–monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design. PMID:18550812

  11. Supramolecular Architecture of the Coronavirus Particle.

    PubMed

    Neuman, B W; Buchmeier, M J

    2016-01-01

    Coronavirus particles serve three fundamentally important functions in infection. The virion provides the means to deliver the viral genome across the plasma membrane of a host cell. The virion is also a means of escape for newly synthesized genomes. Lastly, the virion is a durable vessel that protects the genome on its journey between cells. This review summarizes the available X-ray crystallography, NMR, and cryoelectron microscopy structural data for coronavirus structural proteins, and looks at the role of each of the major structural proteins in virus entry and assembly. The potential wider conservation of the nucleoprotein fold identified in the Arteriviridae and Coronaviridae families and a speculative model for the evolution of corona-like virus architecture are discussed. © 2016 Elsevier Inc. All rights reserved.

  12. Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

    PubMed Central

    Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

    2015-01-01

    ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

  13. Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells.

    PubMed

    Millet, Jean Kaoru; Whittaker, Gary R

    2018-04-01

    During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca 2+ -dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. From SARS coronavirus to novel animal and human coronaviruses.

    PubMed

    To, Kelvin K W; Hung, Ivan F N; Chan, Jasper F W; Yuen, Kwok-Yung

    2013-08-01

    In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) caused one of the most devastating epidemics known to the developed world. There were two important lessons from this epidemic. Firstly, coronaviruses, in addition to influenza viruses, can cause severe and rapidly spreading human infections. Secondly, bats can serve as the origin and natural animal reservoir of deadly human viruses. Since then, researchers around the world, especially those in Asia where SARS-CoV was first identified, have turned their focus to find novel coronaviruses infecting humans, bats, and other animals. Two human coronaviruses, HCoV-HKU1 and HCoV-NL63, were identified shortly after the SARS-CoV epidemic as common causes of human respiratory tract infections. In 2012, a novel human coronavirus, now called Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in the Middle East to cause fatal human infections in three continents. MERS-CoV human infection is similar to SARS-CoV in having a high fatality rate and the ability to spread from person to person which resulted in secondary cases among close contacts including healthcare workers without travel history to the Middle East. Both viruses also have close relationships with bat coronaviruses. New cases of MERS-CoV infection in humans continue to occur with the origins of the virus still unknown in many cases. A multifaceted approach is necessary to control this evolving MERS-CoV outbreak. Source identification requires detailed epidemiological studies of the infected patients and enhanced surveillance of MERS-CoV or similar coronaviruses in humans and animals. Early diagnosis of infected patients and appropriate infection control measures will limit the spread in hospitals, while social distancing strategies may be necessary to control the outbreak in communities if it remained uncontrolled as in the SARS epidemic.

  15. Envelope Protein Dynamics in Paramyxovirus Entry

    PubMed Central

    Plattet, Philippe; Plemper, Richard K.

    2013-01-01

    ABSTRACT Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics. PMID:23820396

  16. Envelope protein dynamics in paramyxovirus entry.

    PubMed

    Plattet, Philippe; Plemper, Richard K

    2013-07-02

    Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics.

  17. Structure of the C-terminal domain of nsp4 from feline coronavirus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes.more » In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.« less

  18. Abelson Kinase Inhibitors Are Potent Inhibitors of Severe Acute Respiratory Syndrome Coronavirus and Middle East Respiratory Syndrome Coronavirus Fusion

    PubMed Central

    Coleman, Christopher M.; Sisk, Jeanne M.; Mingo, Rebecca M.; Nelson, Elizabeth A.; White, Judith M.

    2016-01-01

    ABSTRACT The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause significant morbidity and morality. There is currently no approved therapeutic for highly pathogenic coronaviruses, even as MERS-CoV is spreading throughout the Middle East. We previously screened a library of FDA-approved drugs for inhibitors of coronavirus replication in which we identified Abelson (Abl) kinase inhibitors, including the anticancer drug imatinib, as inhibitors of both SARS-CoV and MERS-CoV in vitro. Here we show that the anti-CoV activity of imatinib occurs at the early stages of infection, after internalization and endosomal trafficking, by inhibiting fusion of the virions at the endosomal membrane. We specifically identified the imatinib target, Abelson tyrosine-protein kinase 2 (Abl2), as required for efficient SARS-CoV and MERS-CoV replication in vitro. These data demonstrate that specific approved drugs can be characterized in vitro for their anticoronavirus activity and used to identify host proteins required for coronavirus replication. This type of study is an important step in the repurposing of approved drugs for treatment of emerging coronaviruses. IMPORTANCE Both SARS-CoV and MERS-CoV are zoonotic infections, with bats as the primary source. The 2003 SARS-CoV outbreak began in Guangdong Province in China and spread to humans via civet cats and raccoon dogs in the wet markets before spreading to 37 countries. The virus caused 8,096 confirmed cases of SARS and 774 deaths (a case fatality rate of ∼10%). The MERS-CoV outbreak began in Saudi Arabia and has spread to 27 countries. MERS-CoV is believed to have emerged from bats and passed into humans via camels. The ongoing outbreak of MERS-CoV has resulted in 1,791 cases of MERS and 640 deaths (a case fatality rate of 36%). The emergence of SARS-CoV and MERS-CoV provides evidence that coronaviruses are currently spreading from zoonotic

  19. Removal of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product quality.

    PubMed

    Rodrigues, Teresa; Alves, Ana; Lopes, António; Carrondo, Manuel J T; Alves, Paula M; Cruz, Pedro E

    2008-10-01

    We have investigated the role of the retroviral lipid bilayer and envelope proteins in the adsorption of retroviral vectors (RVs) to a Fractogel DEAE matrix. Intact RVs and their degradation components (envelope protein-free vectors and solubilized vector components) were adsorbed to this matrix and eluted using a linear gradient. Envelope protein-free RVs (Env(-)) and soluble envelope proteins (gp70) eluted in a significantly lower range of conductivities than intact RVs (Env(+)) (13.7-30 mS/cm for Env(-) and gp70 proteins vs. 47-80 mS/cm for Env(+)). The zeta (zeta)-potential of Env(+) and Env(-) vectors was evaluated showing that envelope proteins define the pI of the viral particles (pI (Env(+)) < 2 versus 3 < pI (Env(-)) < 4) and that Env(+) and Env(-) vectors have similar zeta-potentials within pH 5 and 8. The results presented herein indicate that the adsorption of retroviral particles occurs through multi-point interaction of the envelope proteins with the cationic groups on the chromatographic matrix. The strength of this adsorption is thus dependent on the amount of envelope protein present in the viral lipid bilayer. In conclusion, AEXc enables the separation of gp70 proteins as well as envelope protein-free vectors constituting a significant improvement to the quality of retroviral preparations for gene therapy applications.

  20. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    PubMed Central

    Thor, Sharmi W.; Hilt, Deborah A.; Kissinger, Jessica C.; Paterson, Andrew H.; Jackwood, Mark W.

    2011-01-01

    Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. PMID:21994806

  1. Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

    PubMed Central

    Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

    2010-01-01

    Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

  2. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    PubMed

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus.

    PubMed

    Luo, Chu-Ming; Wang, Ning; Yang, Xing-Lou; Liu, Hai-Zhou; Zhang, Wei; Li, Bei; Hu, Ben; Peng, Cheng; Geng, Qi-Bin; Zhu, Guang-Jian; Li, Fang; Shi, Zheng-Li

    2018-07-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs that belong to the same species as MERS-CoV have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1,059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus coronavirus HKU4 , Pipistrellus pipistrellus coronavirus HKU5 , and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io , from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein-protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs have acquired their spike genes from a DPP4-recognizing bat coronavirus HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV. IMPORTANCE Previous studies suggested that MERS-CoV originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses in eight bat species. We provide evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus, HKU4. Our study expands the host ranges of MERS-related CoV and represents an

  4. SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

    PubMed

    Vasilenko, Natalia; Moshynskyy, Igor; Zakhartchouk, Alexander

    2010-02-09

    The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.

  5. Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2016-01-01

    Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

  6. A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

    PubMed Central

    de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

    2015-01-01

    ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although

  7. Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

    NASA Astrophysics Data System (ADS)

    McReynolds, Susanna; Jiang, Shaokai; Rong, Lijun; Caffrey, Michael

    2009-12-01

    The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141-1193 of S) in the prefusion and newly discovered transition states by NMR 15N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.

  8. Bat-to-human: spike features determining 'host jump' of coronaviruses SARS-CoV, MERS-CoV, and beyond.

    PubMed

    Lu, Guangwen; Wang, Qihui; Gao, George F

    2015-08-01

    Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that crossed the species barriers to infect humans. The mechanism of viral interspecies transmission is an important scientific question to be addressed. These coronaviruses contain a surface-located spike (S) protein that initiates infection by mediating receptor-recognition and membrane fusion and is therefore a key factor in host specificity. In addition, the S protein needs to be cleaved by host proteases before executing fusion, making these proteases a second determinant of coronavirus interspecies infection. Here, we summarize the progress made in the past decade in understanding the cross-species transmission of SARS-CoV and MERS-CoV by focusing on the features of the S protein, its receptor-binding characteristics, and the cleavage process involved in priming. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis.

    PubMed

    Sano, Kaori; Kawaguchi, Mari; Katano, Keita; Tomita, Kenji; Inokuchi, Mayu; Nagasawa, Tatsuki; Hiroi, Junya; Kaneko, Toyoji; Kitagawa, Takashi; Fujimoto, Takafumi; Arai, Katsutoshi; Tanaka, Masaru; Yasumasu, Shigeki

    2017-05-01

    Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin. © 2017 Wiley Periodicals, Inc.

  10. Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

    PubMed

    Lau, Susanna K P; Woo, Patrick C Y; Yip, Cyril C Y; Fan, Rachel Y Y; Huang, Yi; Wang, Ming; Guo, Rongtong; Lam, Carol S F; Tsang, Alan K L; Lai, Kenneth K Y; Chan, Kwok-Hung; Che, Xiao-Yan; Zheng, Bo-Jian; Yuen, Kwok-Yung

    2012-05-01

    We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

  11. From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.

    PubMed

    Hilgenfeld, Rolf; Peiris, Malik

    2013-10-01

    This article introduces a series of invited papers in Antiviral Research marking the 10th anniversary of the outbreak of severe acute respiratory syndrome (SARS), caused by a novel coronavirus that emerged in southern China in late 2002. Until that time, coronaviruses had not been recognized as agents causing severe disease in humans, hence, the emergence of the SARS-CoV came as a complete surprise. Research during the past ten years has revealed the existence of a diverse pool of coronaviruses circulating among various bat species and other animals, suggesting that further introductions of highly pathogenic coronaviruses into the human population are not merely probable, but inevitable. The recent emergence of another coronavirus causing severe disease, Middle East respiratory syndrome (MERS), in humans, has made it clear that coronaviruses pose a major threat to human health, and that more research is urgently needed to elucidate their replication mechanisms, identify potential drug targets, and develop effective countermeasures. In this series, experts in many different aspects of coronavirus replication and disease will provide authoritative, up-to-date reviews of the following topics: - clinical management and infection control of SARS; - reservoir hosts of coronaviruses; - receptor recognition and cross-species transmission of SARS-CoV; - SARS-CoV evasion of innate immune responses; - structures and functions of individual coronaviral proteins; - anti-coronavirus drug discovery and development; and - the public health legacy of the SARS outbreak. Each article will be identified in the last line of its abstract as belonging to the series "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses." Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Receptor recognition and cross-species infections of SARS coronavirus

    PubMed Central

    Li, Fang

    2013-01-01

    Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.” PMID:23994189

  13. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  14. Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus

    PubMed Central

    Woo, Patrick C. Y.; Lau, Susanna K. P.; Lam, Carol S. F.; Lau, Candy C. Y.; Tsang, Alan K. L.; Lau, John H. N.; Bai, Ru; Teng, Jade L. L.; Tsang, Chris C. C.; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung

    2012-01-01

    Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination. PMID:22278237

  15. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Lau, Candy C Y; Tsang, Alan K L; Lau, John H N; Bai, Ru; Teng, Jade L L; Tsang, Chris C C; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2012-04-01

    Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination.

  16. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, Jian; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071; Zhang, Huaidong

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type Imore » viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.« less

  17. Development of Animal Models Against Emerging Coronaviruses: From SARS to MERS coronavirus

    PubMed Central

    Sutton, Troy C; Subbarao, Kanta

    2016-01-01

    Two novel coronaviruses have emerged to cause severe disease in humans. While bats may be the primary reservoir for both viruses, SARS coronavirus (SARS-CoV) likely crossed into humans from civets in China, and MERS coronavirus (MERS-CoV) has been transmitted from camels in the Middle East. Unlike SARS-CoV that resolved within a year, continued introductions of MERS-CoV present an on-going public health threat. Animal models are needed to evaluate countermeasures against emerging viruses. With SARS-CoV, several animal species were permissive to infection. In contrast, most laboratory animals are refractory or only semi-permissive to infection with MERS-CoV. This host-range restriction is largely determined by sequence heterogeneity in the MERS-CoV receptor. We describe animal models developed to study coronaviruses, with a focus on host-range restriction at the level of the viral receptor and discuss approaches to consider in developing a model to evaluate countermeasures against MERS-CoV. PMID:25791336

  18. Emergence of Pathogenic Coronaviruses in Cats by Homologous Recombination between Feline and Canine Coronaviruses

    PubMed Central

    Terada, Yutaka; Matsui, Nobutaka; Noguchi, Keita; Kuwata, Ryusei; Shimoda, Hiroshi; Soma, Takehisa; Mochizuki, Masami; Maeda, Ken

    2014-01-01

    Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently. PMID:25180686

  19. Deltabaculoviruses encode a functional type I budded virus envelope fusion protein

    USDA-ARS?s Scientific Manuscript database

    Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cells in culture. An F protein homolog gene was absent in gammabaculoviruses. Here we show tha...

  20. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  1. Receptor recognition and cross-species infections of SARS coronavirus.

    PubMed

    Li, Fang

    2013-10-01

    Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Proteins required for lipopolysaccharide assembly in Escherichia coli form a trans-envelope complex†

    PubMed Central

    Chng, Shu-Sin; Gronenberg, Luisa S.; Kahne, Daniel

    2010-01-01

    The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope. PMID:20446753

  3. Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus.

    PubMed

    Sevajol, Marion; Subissi, Lorenzo; Decroly, Etienne; Canard, Bruno; Imbert, Isabelle

    2014-12-19

    The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis

    PubMed Central

    Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

    2000-01-01

    Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

  5. Protein targeting and integration signal for the chloroplastic outer envelope membrane.

    PubMed Central

    Li, H M; Chen, L J

    1996-01-01

    Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

  6. Development of animal models against emerging coronaviruses: From SARS to MERS coronavirus.

    PubMed

    Sutton, Troy C; Subbarao, Kanta

    2015-05-01

    Two novel coronaviruses have emerged to cause severe disease in humans. While bats may be the primary reservoir for both viruses, SARS coronavirus (SARS-CoV) likely crossed into humans from civets in China, and MERS coronavirus (MERS-CoV) has been transmitted from camels in the Middle East. Unlike SARS-CoV that resolved within a year, continued introductions of MERS-CoV present an on-going public health threat. Animal models are needed to evaluate countermeasures against emerging viruses. With SARS-CoV, several animal species were permissive to infection. In contrast, most laboratory animals are refractory or only semi-permissive to infection with MERS-CoV. This host-range restriction is largely determined by sequence heterogeneity in the MERS-CoV receptor. We describe animal models developed to study coronaviruses, with a focus on host-range restriction at the level of the viral receptor and discuss approaches to consider in developing a model to evaluate countermeasures against MERS-CoV. Copyright © 2015. Published by Elsevier Inc.

  7. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

    PubMed Central

    Kim, Yunjeong; Liu, Hongwei; Galasiti Kankanamalage, Anushka C.; Weerasekara, Sahani; Hua, Duy H.; Groutas, William C.; Chang, Kyeong-Ok; Pedersen, Niels C.

    2016-01-01

    Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further

  8. [Human coronavirus infections: importance and diagnosis].

    PubMed

    Vabret, A; Brouard, J; Petitjean, J; Eugene-Ruellan, G; Freymuth, F

    1998-11-14

    POORLY-KNOWN VIRUS: Coronaviruses, so named because of their sun-ray-like aspect, were discovered in the sixties. The biology of these RNA viruses is complex and poorly understood. KNOWN PATHOGENS: Coronaviruses are known pathogens in veterinary medicine, causing disease states in several domestic species. In human medicine, they can cause benign respiratory infections, but few laboratories include coronaviruses in their routine diagnostic tests. SUSPECTED PATHOGENS: There is some data in the literature suggesting coronaviruses might be implicated in more severe diseases including multiple sclerosis, necrotizing enterocolitis, and lower respiratory tract infections, particularly in infants. IMPROVING DIAGNOSTIC METHODS: Due to the lack of reliable and sensitive diagnostic techniques, it is impossible to date to correctly assess the medical impact of these ubiquitous and endemic viruses. Molecular biology techniques enabling detection of human coronavirus infections should be applied to verifying the suspected implication of these viruses in diverse disease states.

  9. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

    PubMed

    Endow, Joshua K; Rocha, Agostinho Gomes; Baldwin, Amy J; Roston, Rebecca L; Yamaguchi, Toshio; Kamikubo, Hironari; Inoue, Kentaro

    2016-01-01

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

  10. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

    DOE PAGES

    Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; ...

    2016-12-09

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

  11. Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study.

    PubMed

    Reusken, Chantal B E M; Haagmans, Bart L; Müller, Marcel A; Gutierrez, Carlos; Godeke, Gert-Jan; Meyer, Benjamin; Muth, Doreen; Raj, V Stalin; Smits-De Vries, Laura; Corman, Victor M; Drexler, Jan-Felix; Smits, Saskia L; El Tahir, Yasmin E; De Sousa, Rita; van Beek, Janko; Nowotny, Norbert; van Maanen, Kees; Hidalgo-Hermoso, Ezequiel; Bosch, Berend-Jan; Rottier, Peter; Osterhaus, Albert; Gortázar-Schmidt, Christian; Drosten, Christian; Koopmans, Marion P G

    2013-10-01

    A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    PubMed

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

  13. The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

    PubMed

    Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-10-13

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model.

    PubMed

    de Wilde, Adriaan H; Falzarano, Darryl; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Fett, Craig; Martellaro, Cynthia; Posthuma, Clara C; Feldmann, Heinz; Perlman, Stanley; Snijder, Eric J

    2017-01-15

    Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Molecular basis of endosomal-membrane association for the dengue virus envelope protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

    Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

  16. Molecular basis of endosomal-membrane association for the dengue virus envelope protein

    DOE PAGES

    Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

    2015-01-02

    Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

  17. The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

    PubMed Central

    Friedel, Caroline C.; Müller, Marcel A.; Carbajo-Lozoya, Javier; Stellberger, Thorsten; von Dall’Armi, Ekatarina; Herzog, Petra; Kallies, Stefan; Niemeyer, Daniela; Ditt, Vanessa; Kuri, Thomas; Züst, Roland; Pumpor, Ksenia; Hilgenfeld, Rolf; Schwarz, Frank; Zimmer, Ralf; Steffen, Imke; Weber, Friedemann; Thiel, Volker; Herrler, Georg; Thiel, Heinz-Jürgen; Schwegmann-Weßels, Christel; Pöhlmann, Stefan; Haas, Jürgen; Drosten, Christian; von Brunn, Albrecht

    2011-01-01

    Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock. PMID:22046132

  18. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

    PubMed

    Gennaris, Alexandra; Ezraty, Benjamin; Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-12-17

    The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

  19. Rapid Detection of Infectious Envelope Proteins by Magnetoplasmonic Toroidal Metasensors.

    PubMed

    Ahmadivand, Arash; Gerislioglu, Burak; Manickam, Pandiaraj; Kaushik, Ajeet; Bhansali, Shekhar; Nair, Madhavan; Pala, Nezih

    2017-09-22

    Unconventional characteristics of magnetic toroidal multipoles have triggered researchers to study these unique resonant phenomena by using both 3D and planar resonators under intense radiation. Here, going beyond conventional planar unit cells, we report on the observation of magnetic toroidal modes using artificially engineered multimetallic planar plasmonic resonators. The proposed microstructures consist of iron (Fe) and titanium (Ti) components acting as magnetic resonators and torus, respectively. Our numerical studies and following experimental verifications show that the proposed structures allow for excitation of toroidal dipoles in the terahertz (THz) domain with the experimental Q-factor of ∼18. Taking the advantage of high-Q toroidal line shape and its dependence on the environmental perturbations, we demonstrate that room-temperature toroidal metasurface is a reliable platform for immunosensing applications. As a proof of concept, we utilized our plasmonic metasurface to detect Zika-virus (ZIKV) envelope protein (with diameter of 40 nm) using a specific ZIKV antibody. The sharp toroidal resonant modes of the surface functionalized structures shift as a function of the ZIKV envelope protein for small concentrations (∼pM). The results of sensing experiments reveal rapid, accurate, and quantitative detection of envelope proteins with the limit of detection of ∼24.2 pg/mL and sensitivity of 6.47 GHz/log(pg/mL). We envision that the proposed toroidal metasurface opens new avenues for developing low-cost, and efficient THz plasmonic sensors for infection and targeted bioagent detection.

  20. Interferon-Beta 1a and SARS Coronavirus Replication

    DTIC Science & Technology

    2004-02-01

    A global outbreak of severe acute respiratory syn- drome ( SARS ) caused by a novel coronavirus began in March 2003. The rapid emergence of SARS and...emerging infectious disease. The etiologic agent was identified as a coronavirus ( SARS -CoV) that is not closely related to any of the previously...some coronaviruses , including avian infectious bronchitis virus, murine hepati- tis virus, and human coronavirus 229E, are susceptible to type I

  1. Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

    PubMed Central

    Huang, I-Chueh; Bailey, Charles C.; Weyer, Jessica L.; Radoshitzky, Sheli R.; Becker, Michelle M.; Chiang, Jessica J.; Brass, Abraham L.; Ahmed, Asim A.; Chi, Xiaoli; Dong, Lian; Longobardi, Lindsay E.; Boltz, Dutch; Kuhn, Jens H.; Elledge, Stephen J.; Bavari, Sina; Denison, Mark R.; Choe, Hyeryun; Farzan, Michael

    2011-01-01

    Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP1,2) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression. PMID:21253575

  2. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

    PubMed Central

    Le Poder, Sophie

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP) and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV) will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses. PMID:22312347

  3. Feline and canine coronaviruses: common genetic and pathobiological features.

    PubMed

    Le Poder, Sophie

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP) and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV) will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses.

  4. Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History

    PubMed Central

    Tao, Ying; Shi, Mang; Chommanard, Christina; Queen, Krista; Zhang, Jing; Markotter, Wanda; Kuzmin, Ivan V.; Holmes, Edward C.

    2017-01-01

    ABSTRACT Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. PMID:28077633

  5. Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

    PubMed Central

    Denner, Joachim

    2013-01-01

    The induction of neutralizing antibodies is a promising way to prevent retrovirus infections. Neutralizing antibodies are mainly directed against the envelope proteins, which consist of two molecules, the surface envelope (SU) protein and the transmembrane envelope (TM) protein. Antibodies broadly neutralizing the human immunodeficiencvy virus-1 (HIV-1) and binding to the TM protein gp41 of the virus have been isolated from infected individuals. Their epitopes are located in the membrane proximal external region (MPER). Since there are difficulties to induce such neutralizing antibodies as basis for an effective AIDS vaccine, we performed a comparative analysis immunising with the TM proteins of different viruses from the family Retroviridae. Both subfamilies, the Orthoretrovirinae and the Spumaretrovirinae were included. In this study, the TM proteins of three gammaretroviruses including (1) the porcine endogenous retrovirus (PERV), (2) the Koala retrovirus (KoRV), (3) the feline leukemia virus (FeLV), of two lentiviruses, HIV-1, HIV-2, and of two spumaviruses, the feline foamy virus (FFV) and the primate foamy virus (PFV) were used for immunisation. Whereas in all immunisation studies binding antibodies were induced, neutralizing antibodies were only found in the case of the gammaretroviruses. The induced antibodies were directed against the MPER and the fusion peptide proximal region (FPPR) of their TM proteins; however only the antibodies against the MPER were neutralizing. Most importantly, the epitopes in the MPER were localized in the same position as the epitopes of the antibodies broadly neutralizing HIV-1 in the TM protein gp41 of HIV-1, indicating that the MPER is an effective target for the neutralization of retroviruses. PMID:23249763

  6. Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

    PubMed

    Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

    2017-08-19

    Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Data on the association of the nuclear envelope protein Sun1 with nucleoli.

    PubMed

    Moujaber, Ossama; Omran, Nawal; Kodiha, Mohamed; Pié, Brigitte; Cooper, Ellis; Presley, John F; Stochaj, Ursula

    2017-08-01

    SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.

  8. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

    PubMed Central

    Lau, Susanna K. P.; Feng, Yun; Chen, Honglin; Luk, Hayes K. H.; Yang, Wei-Hong; Li, Kenneth S. M.; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y. Y.; Ahmed, Syed Shakeel; Yeung, Hazel C.; Lam, Carol S. F.; Cai, Jian-Piao; Wong, Samson S. Y.; Chan, Jasper F. W.; Yuen, Kwok-Yung

    2015-01-01

    ABSTRACT Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS

  9. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination.

    PubMed

    Lau, Susanna K P; Feng, Yun; Chen, Honglin; Luk, Hayes K H; Yang, Wei-Hong; Li, Kenneth S M; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y Y; Ahmed, Syed Shakeel; Yeung, Hazel C; Lam, Carol S F; Cai, Jian-Piao; Wong, Samson S Y; Chan, Jasper F W; Yuen, Kwok-Yung; Zhang, Hai-Lin; Woo, Patrick C Y

    2015-10-01

    Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8

  10. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

    PubMed Central

    Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-01-01

    The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts 1-3. Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both R- and S- diastereoisomers of Met-O, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting Met residues from oxidation should prompt search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum (ER). PMID:26641313

  11. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2014-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2′ position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2′ site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus. PMID:25288733

  12. [Development of a SPA-ELISA method for detecting anti-coronavirus IgG antibodies in serum samples from fulvous fruit bats].

    PubMed

    Zhou, Jie; Liao, Yu-xue; Chen, Zhong; Li, Yu-chun; Gao, Lu-Lu; Chen, Yi-xiong; Cai, Lian-gong; Chen, Qing; Yu, Shou-yi

    2008-05-01

    To develop an simple and sensitive method for detecting anti-coronavirus IgG antibodies in bat sera based on enzyme-linked immunosorbent assay (ELISA). A commercial ELISA kit for detecting SARS-CoV antibody was modified for detecting coronavirus antibodies in bat serum samples. The second antibody in the kit was replaced with horseradish peroxidase-conjugated protein-A (HRP-SPA) based on the characteristics of binding between Staphylococcus aureus protein A (SPA) and mammal IgG Fc fragment. The sera of 55 fulvous fruit bats (Rousettus dasymallus) were tested using the SPA-ELISA. The test results of the positive and negative controls in the kit and the serum samples from convalescent ;patient were consistent with expectation. Coronavirus antibody was detected in 2 out of the 55 bat serum samples. Serum neutralization test confirmed the validity of the SPA-ELISA method. This SPA-ELISA method is applicable for detecting coronavirus antibody in bat sera.

  13. Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Tsang, Alan K L; Hui, Suk-Wai; Fan, Rachel Y Y; Martelli, Paolo; Yuen, Kwok-Yung

    2014-01-01

    While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 10(3) to 1 × 10(5) copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

  14. Detection of group 1 coronaviruses in bats in North America

    USGS Publications Warehouse

    Dominguez, S.R.; O'Shea, T.J.; Oko, L.M.; Holmes, K.V.

    2007-01-01

    The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People's Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription-PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

  15. Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

    PubMed Central

    Suomalainen, M; Garoff, H

    1994-01-01

    The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways. Images PMID:8035486

  16. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulatedmore » strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.« less

  17. Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

    PubMed Central

    Honda-Okubo, Yoshikazu; Barnard, Dale; Ong, Chun Hao; Peng, Bi-Hung; Tseng, Chien-Te Kent

    2014-01-01

    ABSTRACT Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. IMPORTANCE Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified

  18. Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

    PubMed Central

    Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

    2010-01-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

  19. A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

    PubMed Central

    Westerbeck, Jason W.

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the

  20. Coronavirus infections in horses in Saudi Arabia and Oman.

    PubMed

    Hemida, M G; Chu, D K W; Perera, R A P M; Ko, R L W; So, R T Y; Ng, B C Y; Chan, S M S; Chu, S; Alnaeem, A A; Alhammadi, M A; Webby, R J; Poon, L L M; Balasuriya, U B R; Peiris, M

    2017-12-01

    Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT-PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT-PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross-reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. © 2017 Blackwell Verlag GmbH.

  1. Crystal structure of the Japanese encephalitis virus envelope protein.

    PubMed

    Luca, Vincent C; AbiMansour, Jad; Nelson, Christopher A; Fremont, Daved H

    2012-02-01

    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

  2. Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

    PubMed Central

    Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

    2009-01-01

    The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the

  3. Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

    PubMed

    Lee, Saemi; Jo, Seong-Deok; Son, Kidong; An, Injung; Jeong, Jipseol; Wang, Seung-Jun; Kim, Yongkwan; Jheong, Weonhwa; Oem, Jae-Ku

    2018-01-01

    Bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (SARS), coronavirus, and other coronaviruses found in mammals. However, little research has been conducted on bat coronaviruses in South Korea. In this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in South Korea. RT-PCR and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. Coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family Rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family Vespertiliodae. To determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the RNA-dependent RNA polymerase (RdRp) gene were analyzed phylogenetically. Thirteen sequences belonging to SARS-like betacoronaviruses showed the highest nucleotide identity (97.1-99.7%) with Bat-CoV-JTMC15 reported in China. The other five sequences were most similar to MERS-like betacoronaviruses. Four nucleotide sequences displayed the highest identity (94.1-95.1%) with Bat-CoV-HKU5 from Hong Kong. The one sequence from a carcass showed the highest nucleotide identity (99%) with Bat-CoV-SC2013 from China. These results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs.

  4. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

    PubMed

    Ge, Xing-Yi; Li, Jia-Lu; Yang, Xing-Lou; Chmura, Aleksei A; Zhu, Guangjian; Epstein, Jonathan H; Mazet, Jonna K; Hu, Ben; Zhang, Wei; Peng, Cheng; Zhang, Yu-Ji; Luo, Chu-Ming; Tan, Bing; Wang, Ning; Zhu, Yan; Crameri, Gary; Zhang, Shu-Yi; Wang, Lin-Fa; Daszak, Peter; Shi, Zheng-Li

    2013-11-28

    The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

  5. Understanding bat SARS-like coronaviruses for the preparation of future coronavirus outbreaks - Implications for coronavirus vaccine development.

    PubMed

    Ng, Oi-Wing; Tan, Yee-Joo

    2017-01-02

    The severe acute respiratory syndrome coronavirus (SARS-CoV) first emerged in 2003, causing the SARS epidemic which resulted in a 10% fatality rate. The advancements in metagenomic techniques have allowed the identification of SARS-like coronaviruses (SL-CoVs) sequences that share high homology to the human SARS-CoV epidemic strains from wildlife bats, presenting concrete evidence that bats are the origin and natural reservoir of SARS-CoV. The application of reverse genetics further enabled that characterization of these bat CoVs and the prediction of their potential to cause disease in humans. The knowledge gained from such studies is valuable in the surveillance and preparation of a possible future outbreak caused by a spill-over of these bat SL-CoVs.

  6. Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein.

    PubMed

    Chen, Huiqing; Li, Mei; Huang, Guoping; Mai, Weijun; Chen, Keping; Zhou, Yajing

    2014-08-01

    Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

  7. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  8. Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang,X.; Wu, J.; Sivaraman, J.

    2007-01-01

    White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 {angstrom}, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelopemore » proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt {beta}-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core {beta}-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.« less

  9. Coronavirus 229E-related pneumonia in immunocompromised patients.

    PubMed

    Pene, Frédéric; Merlat, Annabelle; Vabret, Astrid; Rozenberg, Flore; Buzyn, Agnès; Dreyfus, François; Cariou, Alain; Freymuth, François; Lebon, Pierre

    2003-10-01

    Coronaviruses strains 229E and OC43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. Although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. Here we report 2 well-documented cases of pneumonia related to coronavirus 229E, each with a different clinical presentation. Diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase-polymerase chain reaction. On the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients.

  10. Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

    PubMed Central

    Bohannon, Kevin Patrick; Jun, Yonggun; Gross, Steven P.; Smith, Gregory Allan

    2013-01-01

    The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection. PMID:23569236

  11. Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture.

    PubMed

    Bohannon, Kevin Patrick; Jun, Yonggun; Gross, Steven P; Smith, Gregory Allan

    2013-04-23

    The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

  12. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    USDA-ARS?s Scientific Manuscript database

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  13. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    PubMed

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  14. Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

    PubMed

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2017-02-01

    Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

  15. Crystal Structure of the Japanese Encephalitis Virus Envelope Protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luca, Vincent C.; AbiMansour, Jad; Nelson, Christopher A.

    2012-03-13

    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-{angstrom} resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimermore » in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.« less

  16. OOCYTE ENVELOPE PROTEINS AND VITELLOGENIN IN MALE SHEEPHEAD MINNOW EXPOSED TO ESTRADIOL

    EPA Science Inventory

    Oocyte Envelope Proteins and Vitellogenin Expression in Male Sheepshead Minnows Exposed to Estradiol (Abstract). To be presented at the 22nd Annual Meeting of the Society of Environmental Toxicology and Chemistry: Changing Environmental Awareness: Societal Concerns and Scientifi...

  17. Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

    PubMed Central

    Zattas, Dimitrios; Hochstrasser, Mark

    2014-01-01

    The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

  18. Coronavirus Infection and Diversity in Bats in the Australasian Region.

    PubMed

    Smith, C S; de Jong, C E; Meers, J; Henning, J; Wang, L- F; Field, H E

    2016-03-01

    Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

  19. The SARS-Coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

    PubMed Central

    Baez-Santos, Yahira M.; St. John, Sarah E.; Mesecar, Andrew D.

    2018-01-01

    Over ten years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8,500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series

  20. SARS and MERS: recent insights into emerging coronaviruses.

    PubMed

    de Wit, Emmie; van Doremalen, Neeltje; Falzarano, Darryl; Munster, Vincent J

    2016-08-01

    The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The continuing introductions of MERS-CoV from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. Scientific advancements since the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of MERS-CoV and the development of therapeutics. In this Review, we detail our present understanding of the transmission and pathogenesis of SARS-CoV and MERS-CoV, and discuss the current state of development of measures to combat emerging coronaviruses.

  1. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ostlund, Cecilia; Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032; Guan, Tinglu

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients withmore » FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.« less

  2. Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

    PubMed Central

    Navarre, William Wiley; Schneewind, Olaf

    1999-01-01

    The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

  3. A molecular arms race between host innate antiviral response and emerging human coronaviruses.

    PubMed

    Wong, Lok-Yin Roy; Lui, Pak-Yin; Jin, Dong-Yan

    2016-02-01

    Coronaviruses have been closely related with mankind for thousands of years. Community-acquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

  4. T-cell-mediated immune response to respiratory coronaviruses

    PubMed Central

    Channappanavar, Rudragouda; Zhao, Jincun; Perlman, Stanley

    2014-01-01

    Emerging respiratory coronaviruses such as the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) pose potential biological threats to humans. SARS and MERS are manifested as severe atypical pneumonia associated with high morbidity and mortality in humans. The majority of studies carried out in SARS-CoV-infected humans and animals attribute a dysregulated/exuberant innate response as a leading contributor to SARS-CoV-mediated pathology. A decade after the 2002–2003 SARS epidemic, we do not have any approved preventive or therapeutic agents available in case of re-emergence of SARS-CoV or other related viruses. A strong neutralizing antibody response generated against the spike (S) glycoprotein of SARS-CoV is completely protective in the susceptible host. However, neutralizing antibody titers and the memory B cell response are short-lived in SARS-recovered patients and the antibody will target primary homologous strain. Interestingly, the acute phase of SARS in humans is associated with a severe reduction in the number of T cells in the blood. Surprisingly, only a limited number of studies have explored the role of the T cell-mediated adaptive immune response in respiratory coronavirus pathogenesis. In this review, we discuss the role of anti-virus CD4 and CD8 T cells during respiratory coronavirus infections with a special emphasis on emerging coronaviruses. PMID:24845462

  5. The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation.

    PubMed

    Bermeo, Sandra; Al-Saedi, Ahmed; Kassem, Moustapha; Vidal, Christopher; Duque, Gustavo

    2017-12-01

    Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro-osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic differentiation were performed. MAN1 knockdown increased osteogenesis and mineralization. In contrast, osteogenesis remained stable upon MAN1 overexpression. Regarding a mechanism, we found that low levels of MAN1 facilitated the nuclear accumulation of regulatory smads and smads-related complexes, with a concurrently high expression of nuclear β-Catenin. In addition, we found adipogenesis to be decreased in both conditions, although predominantly affected by MAN1 overexpression. Finally, lamin A, a protein of the nuclear envelope that regulates MSC differentiation, was unaffected by changes in MAN1. In conclusion, our studies demonstrated that lower levels of MAN1 in differentiating MSC are associated with higher osteogenesis and lower adipogenesis. High levels of MAN1 only affected adipogenesis. These effects could have an important role in the understanding of the role of the proteins of the nuclear envelope in bone formation. J. Cell. Biochem. 118: 4425-4435, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. Proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo

    PubMed Central

    2012-01-01

    Background Infectious bronchitis virus (IBV) is first to be discovered coronavirus which is probably endemic in all regions with intensive impact on poultry production. In this study, we used two-dimensional gel electrophoresis (2-DE) and two-dimensional fluorescence difference gel electrophoresis (2-DIGE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), to explore the global proteome profiles of trachea and kidney tissues from chicken at different stages infected in vivo with the highly virulent ck/CH/LDL/97I P5 strain of infectious bronchitis virus (IBV) and the embryo-passaged, attenuated ck/CH/LDL/97I P115 strain. Results Fifty-eight differentially expressed proteins were identified. Results demonstrated that some proteins which had functions in cytoskeleton organization, anti-oxidative stress, and stress response, showed different change patterns in abundance from chicken infected with the highly virulent ck/CH/LDL/97I P5 strain and those given the embryo-passaged, attenuated P115 stain. In addition, the dynamic transcriptional alterations of 12 selected proteins were analyzed by the real-time RT-PCR, and western blot analysis confirmed the change in abundance of heat shock proteins (HSP) beta-1, annexin A2, and annexin A5. Conclusions The proteomic alterations described here may suggest that these changes to protein expression correlate with IBV virus' virulence in chicken, hence provides valuable insights into the interactions of IBV with its host and may also assist with investigations of the pathogenesis of IBV and other coronavirus infections. PMID:22463732

  7. Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

    PubMed

    Dietzel, Erik; Anderson, Danielle E; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

    2011-07-01

    In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

  8. A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

    PubMed

    Lee, S Y; Poloumienko, A; Belfry, S; Qu, X; Chen, W; MacAfee, N; Morin, B; Lucarotti, C; Krause, M

    1996-01-01

    The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

  9. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection.

    PubMed

    Ng, Oi-Wing; Chia, Adeline; Tan, Anthony T; Jadi, Ramesh S; Leong, Hoe Nam; Bertoletti, Antonio; Tan, Yee-Joo

    2016-04-12

    Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8(+) T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8(+) T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Dynamic nuclear envelope phenotype in rats overexpressing mutated human torsinA protein.

    PubMed

    Yu-Taeger, Libo; Gaiser, Viktoria; Lotzer, Larissa; Roenisch, Tina; Fabry, Benedikt Timo; Stricker-Shaver, Janice; Casadei, Nicolas; Walter, Michael; Schaller, Martin; Riess, Olaf; Nguyen, Huu Phuc; Ott, Thomas; Grundmann-Hauser, Kathrin

    2018-05-08

    A three-base-pair deletion in the human TOR1A gene is causative for the most common form of primary dystonia, the early-onset dystonia type 1 (DYT1 dystonia). The pathophysiological consequences of this mutation are still unknown.To study the pathology of the mutant torsinA (TOR1A) protein, we have generated a transgenic rat line that overexpresses the human mutant protein under the control of the human TOR1A promoter. This new animal model was phenotyped with several approaches, including behavioral tests and neuropathological analyses. A motor phenotype and cellular and ultrastructural key features of torsinA pathology were found in this new transgenic rat line supporting that it can be used as a model system for investigating the disease development. Analyses of mutant TOR1A protein expression in various brain regions also showed a dynamic expression pattern and a reversible nuclear envelope pathology. These findings suggest the differential vulnerabilities of distinct neuronal subpopulations. Furthermore the reversibility of the nuclear envelope pathology might be a therapeutic target to treat the disease. © 2018. Published by The Company of Biologists Ltd.

  11. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

    PubMed

    Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

    2012-08-01

    Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    PubMed

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

  13. Inhibitors of SARS-CoV Entry - Identification using an Internally-Controlled Dual Envelope Pseudovirion Assay

    PubMed Central

    Zhou, Yanchen; Agudelo, Juliet; Lu, Kai; Goetz, David H.; Hansell, Elizabeth; Chen, Yen Ting; Roush, William R.; McKerrow, James; Craik, Charles S.; Amberg, Sean M.; Simmons, Graham

    2011-01-01

    Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, Dual Envelope Pseudovirion (DEP) Assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes. PMID:21820471

  14. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  15. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Changhee; Yoo, Dongwan

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-{delta}E-transfected cells howevermore » produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-{delta}E virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-{delta}E virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm.« less

  16. Plasticity of a critical antigenic determinant in the West Nile virus NY99 envelope protein domain III.

    PubMed

    Plante, Jessica A; Torres, Maricela; Huang, Claire Y-H; Beasley, David W C

    2016-09-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that causes febrile illness, encephalitis, and occasionally death in humans. The envelope protein is the main component of the WNV virion surface, and domain III of the envelope protein (EIII) is both a putative receptor binding domain and a target of highly specific, potently neutralizing antibodies. Envelope E-332 (E-332) is known to have naturally occurring variation and to be a key determinant of neutralization for anti-EIII antibodies. A panel of viruses containing all possible amino acid substitutions at E-332 was constructed. E-332 was found to be highly tolerant of mutation, and almost all of these changes had large impacts on antigenicity of EIII but only limited effects on growth or virulence phenotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  18. Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

    PubMed Central

    Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.; Dinnon, Kenneth H.; Leist, Sarah R.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Stratton, Kelly G.; Cockrell, Adam S.; Debbink, Kari; Sims, Amy C.; Waters, Katrina M.

    2017-01-01

    ABSTRACT Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2′O-methyltransferase (2′O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2′O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2′O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting. IMPORTANCE Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2′O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN

  19. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice

    PubMed Central

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  20. Molecular pathology of emerging coronavirus infections

    PubMed Central

    Gralinski, Lisa E; Baric, Ralph S

    2015-01-01

    Respiratory viruses can cause a wide spectrum of pulmonary diseases, ranging from mild, upper respiratory tract infections to severe and life-threatening lower respiratory tract infections, including the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Viral clearance and subsequent recovery from infection require activation of an effective host immune response; however, many immune effector cells may also cause injury to host tissues. Severe acute respiratory syndrome (SARS) coronavirus and Middle East respiratory syndrome (MERS) coronavirus cause severe infection of the lower respiratory tract, with 10% and 35% overall mortality rates, respectively; however, >50% mortality rates are seen in the aged and immunosuppressed populations. While these viruses are susceptible to interferon treatment in vitro, they both encode numerous genes that allow for successful evasion of the host immune system until after high virus titres have been achieved. In this review, we discuss the importance of the innate immune response and the development of lung pathology following human coronavirus infection. PMID:25270030

  1. Recent Transmission of a Novel Alphacoronavirus, Bat Coronavirus HKU10, from Leschenault's Rousettes to Pomona Leaf-Nosed Bats: First Evidence of Interspecies Transmission of Coronavirus between Bats of Different Suborders

    PubMed Central

    Lau, Susanna K. P.; Li, Kenneth S. M.; Tsang, Alan K. L.; Shek, Chung-Tong; Wang, Ming; Choi, Garnet K. Y.; Guo, Rongtong; Wong, Beatrice H. L.; Poon, Rosana W. S.; Lam, Carol S. F.; Wang, Sylvia Y. H.; Fan, Rachel Y. Y.; Chan, Kwok-Hung; Zheng, Bo-Jian

    2012-01-01

    Although coronaviruses are known to infect various animals by adapting to new hosts, interspecies transmission events are still poorly understood. During a surveillance study from 2005 to 2010, a novel alphacoronavirus, BatCoV HKU10, was detected in two very different bat species, Ro-BatCoV HKU10 in Leschenault's rousettes (Rousettus leschenaulti) (fruit bats in the suborder Megachiroptera) in Guangdong and Hi-BatCoV HKU10 in Pomona leaf-nosed bats (Hipposideros pomona) (insectivorous bats in the suborder Microchiroptera) in Hong Kong. Although infected bats appeared to be healthy, Pomona leaf-nosed bats carrying Hi-BatCoV HKU10 had lower body weights than uninfected bats. To investigate possible interspecies transmission between the two bat species, the complete genomes of two Ro-BatCoV HKU10 and six Hi-BatCoV HKU10 strains were sequenced. Genome and phylogenetic analyses showed that Ro-BatCoV HKU10 and Hi-BatCoV HKU10 represented a novel alphacoronavirus species, sharing highly similar genomes except in the genes encoding spike proteins, which had only 60.5% amino acid identities. Evolution of the spike protein was also rapid in Hi-BatCoV HKU10 strains from 2005 to 2006 but stabilized thereafter. Molecular-clock analysis dated the most recent common ancestor of all BatCoV HKU10 strains to 1959 (highest posterior density regions at 95% [HPDs], 1886 to 2002) and that of Hi-BatCoV HKU10 to 1986 (HPDs, 1956 to 2004). The data suggested recent interspecies transmission from Leschenault's rousettes to Pomona leaf-nosed bats in southern China. Notably, the rapid adaptive genetic change in BatCoV HKU10 spike protein by ∼40% amino acid divergence after recent interspecies transmission was even greater than the ∼20% amino acid divergence between spike proteins of severe acute respiratory syndrome-related Rhinolophus bat coronavirus (SARSr-CoV) in bats and civets. This study provided the first evidence for interspecies transmission of coronavirus between bats of

  2. Detection and Phylogenetic Analysis of Group 1 Coronaviruses in South American Bats

    PubMed Central

    Foster, Jerome E.; Zhu, Hua Chen; Zhang, Jin Xia; Smith, Gavin J.D.; Thompson, Nadin; Auguste, Albert J.; Ramkissoon, Vernie; Adesiyun, Abiodun A.; Guan, Yi

    2008-01-01

    Bat coronaviruses (Bt-CoVs) are thought to be the precursors of severe acute respiratory syndrome coronavirus. We detected Bt-CoVs in 2 bat species from Trinidad. Phylogenetic analysis of the RNA-dependent RNA polymerase gene and helicase confirmed them as group 1 coronaviruses. PMID:19046513

  3. Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein.

    PubMed

    Escriou, Nicolas; Callendret, Benoît; Lorin, Valérie; Combredet, Chantal; Marianneau, Philippe; Février, Michèle; Tangy, Frédéric

    2014-03-01

    The recent identification of a novel human coronavirus responsible of a SARS-like illness in the Middle-East a decade after the SARS pandemic, demonstrates that reemergence of a SARS-like coronavirus from an animal reservoir remains a credible threat. Because SARS is contracted by aerosolized contamination of the respiratory tract, a vaccine inducing mucosal long-term protection would be an asset to control new epidemics. To this aim, we generated live attenuated recombinant measles vaccine (MV) candidates expressing either the membrane-anchored SARS-CoV spike (S) protein or its secreted soluble ectodomain (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S induced the highest titers of neutralizing antibodies and fully protected immunized animals from intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted recombinant Ssol protein, recombinant MV induced stronger and Th1-biased responses, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Gene Targeting of Envoplakin, a Cytoskeletal Linker Protein and Precursor of the Epidermal Cornified Envelope

    PubMed Central

    Määttä, Arto; DiColandrea, Teresa; Groot, Karen; Watt, Fiona M.

    2001-01-01

    Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function. PMID:11564887

  5. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    PubMed Central

    Lahaye, Xavier; Satoh, Takeshi; Gentili, Matteo; Cerboni, Silvia; Silvin, Aymeric; Conrad, Cécile; Ahmed-Belkacem, Abdelhakim; Rodriguez, Elisa C.; Guichou, Jean-François; Bosquet, Nathalie; Piel, Matthieu; Le Grand, Roger; King, Megan C.; Pawlotsky, Jean-Michel; Manel, Nicolas

    2016-01-01

    Summary During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA) is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope. PMID:27149839

  6. Antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine.

    PubMed

    Nemoto, Manabu; Kanno, Toru; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kokado, Hiroshi

    2017-11-17

    A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future.

  7. The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9).

    PubMed

    Chiu, Mei-Wui; Shih, Hsiu-Ming; Yang, Tsung-Han; Yang, Yun-Liang

    2007-05-01

    Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based "Functional Yeast Array". Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.

  8. Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins.

    PubMed

    Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J

    2015-12-30

    Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines

  9. Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

    PubMed

    Lacroix, Audrey; Duong, Veasna; Hul, Vibol; San, Sorn; Davun, Hull; Omaliss, Keo; Chea, Sokha; Hassanin, Alexandre; Theppangna, Watthana; Silithammavong, Soubanh; Khammavong, Kongsy; Singhalath, Sinpakone; Greatorex, Zoe; Fine, Amanda E; Goldstein, Tracey; Olson, Sarah; Joly, Damien O; Keatts, Lucy; Dussart, Philippe; Afelt, Aneta; Frutos, Roger; Buchy, Philippe

    2017-03-01

    South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (βCoV), respectively. The βCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat βCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of βCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    PubMed Central

    Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

  11. FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

    DOE PAGES

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; ...

    2015-05-01

    Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is amore » paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.« less

  12. Membrane vesiculation induced by proteins of the dengue virus envelope studied by molecular dynamics simulations.

    PubMed

    de Oliveira Dos Santos Soares, Ricardo; Bortot, Leandro Oliveira; van der Spoel, David; Caliri, Antonio

    2017-12-20

    Biological membranes are continuously remodeled in the cell by specific membrane-shaping machineries to form, for example, tubes and vesicles. We examine fundamental mechanisms involved in the vesiculation processes induced by a cluster of envelope (E) and membrane (M) proteins of the dengue virus (DENV) using molecular dynamics simulations and a coarse-grained model. We show that an arrangement of three E-M heterotetramers (EM 3 ) works as a bending unit and an ordered cluster of five such units generates a closed vesicle, reminiscent of the virus budding process. In silico mutagenesis of two charged residues of the anchor helices of the envelope proteins of DENV shows that Arg-471 and Arg-60 are fundamental to produce bending stress on the membrane. The fine-tuning between the size of the EM 3 unit and its specific bending action suggests this protein unit is an important factor in determining the viral particle size.

  13. Membrane vesiculation induced by proteins of the dengue virus envelope studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    de Oliveira dos Santos Soares, Ricardo; Oliveira Bortot, Leandro; van der Spoel, David; Caliri, Antonio

    2017-12-01

    Biological membranes are continuously remodeled in the cell by specific membrane-shaping machineries to form, for example, tubes and vesicles. We examine fundamental mechanisms involved in the vesiculation processes induced by a cluster of envelope (E) and membrane (M) proteins of the dengue virus (DENV) using molecular dynamics simulations and a coarse-grained model. We show that an arrangement of three E-M heterotetramers (EM3) works as a bending unit and an ordered cluster of five such units generates a closed vesicle, reminiscent of the virus budding process. In silico mutagenesis of two charged residues of the anchor helices of the envelope proteins of DENV shows that Arg-471 and Arg-60 are fundamental to produce bending stress on the membrane. The fine-tuning between the size of the EM3 unit and its specific bending action suggests this protein unit is an important factor in determining the viral particle size.

  14. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maric, Martina; Haugo, Alison C.; Dauer, William

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but ismore » enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.« less

  15. Murine Sarcoma Virus Gene Expression: Transformants Which Express Viral Envelope Glycoprotein In The Absence Of The Major Internal Protein And Infectious Particles

    PubMed Central

    Bilello, John A.; Strand, Mette; August, J. T.

    1974-01-01

    Expression of the major internal protein and the envelope glycoprotein of murine C-type viruses in focus-derived lines of normal rat kidney cells infected with Kirsten murine sarcoma virus was measured by radioimmunoassay. Of the clones selected, which do not produce virus particles or the major viral structural protein, approximately half express the viral envelope glycoprotein at concentrations found in productively infected cells. Expression of the envelope glycoprotein did not appear to alter significantly the properties of the transformed cells in culture. PMID:4370209

  16. Detection of feline coronavirus using microcantilever sensors

    NASA Astrophysics Data System (ADS)

    Velanki, Sreepriya; Ji, Hai-Feng

    2006-11-01

    This work demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (SARS-CoV) using microcantilever technology by showing that the feline coronavirus (FIP) type I virus can be detected by a microcantilever modified by feline coronavirus (FIP) type I anti-viral antiserum. A microcantilever modified by FIP type I anti-viral antiserum was developed for the detection of FIP type I virus. When the FIP type I virus positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the FIP type I virus by the antiserum on the surface of the microcantilever. A negative control sample that does not contain FIP type I virus did not cause any bending of the microcantilever. The detection limit of the sensor was 0.1 µg ml-1 when the assay time was <1 h.

  17. Identification of an Envelope Protein from the FRD Family of Human Endogenous Retroviruses (HERV-FRD) Conferring Infectivity and Functional Conservation among Simians

    PubMed Central

    Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

    2004-01-01

    A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians—from New World monkeys to humans—are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation—located in the TM subunit—was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution. PMID:14694139

  18. Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians.

    PubMed

    Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

    2004-01-01

    A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.

  19. Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

    PubMed

    Combe, Maxime; Lacoux, Xavier; Martinez, Jérôme; Méjan, Odile; Luciani, Françoise; Daniel, Soizic

    2017-05-01

    Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

    PubMed

    Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

    2009-12-01

    Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

  1. Phylogenetic analysis of the envelope protein (domain lll) of dengue 4 viruses

    PubMed Central

    Mota, Javier; Ramos-Castañeda, José; Rico-Hesse, Rebeca; Ramos, Celso

    2011-01-01

    Objective To evaluate the genetic variability of domain III of envelope (E) protein and to estimate phylogenetic relationships of dengue 4 (Den-4) viruses isolated in Mexico and from other endemic areas of the world. Material and Methods A phylogenetic study of domain III of envelope (E) protein of Den-4 viruses was conducted in 1998 using virus strains from Mexico and other parts of the world, isolated in different years. Specific primers were used to amplify by RT-PCR the domain III and to obtain nucleotide sequence. Based on nucleotide and deduced aminoacid sequence, genetic variability was estimated and a phylogenetic tree was generated. To make an easy genetic analysis of domain III region, a Restriction Fragment Length Polymorphism (RFLP) assay was performed, using six restriction enzymes. Results Study results demonstrate that nucleotide and aminoacid sequence analysis of domain III are similar to those reported from the complete E protein gene. Based on the RFLP analysis of domain III using the restriction enzymes Nla III, Dde I and Cfo I, Den-4 viruses included in this study were clustered into genotypes 1 and 2 previously reported. Conclusions Study results suggest that domain III may be used as a genetic marker for phylogenetic and molecular epidemiology studies of dengue viruses. The English version of this paper is available too at: http://www.insp.mx/salud/index.html PMID:12132320

  2. Antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine

    PubMed Central

    NEMOTO, Manabu; KANNO, Toru; BANNAI, Hiroshi; TSUJIMURA, Koji; YAMANAKA, Takashi; KOKADO, Hiroshi

    2017-01-01

    A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future. PMID:28993568

  3. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    PubMed

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  4. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

    USDA-ARS?s Scientific Manuscript database

    Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of...

  5. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

    PubMed Central

    Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

    2015-01-01

    Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

  6. BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Shiu-Mei; Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan; Huang, Kuo-Jung

    2014-01-20

    Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cellmore » surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.« less

  7. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  8. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

    PubMed

    Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

    2015-06-01

    Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59.

    PubMed

    Li, Pei; Shan, Yiwei; Zheng, Wangliang; Ou, Xiuyuan; Mi, Dan; Mu, Zhixia; Holmes, Kathryn V; Qian, Zhaohui

    2018-06-01

    The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness. IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly

  10. Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

    PubMed

    Fiebig, Uwe; Dieckhoff, Britta; Wurzbacher, Christian; Möller, Annekathrin; Kurth, Reinhard; Denner, Joachim

    2015-04-30

    The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

  11. AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

    PubMed

    Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

    2010-06-01

    Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further

  12. Antigenic regions within the hepatitis C virus envelope 1 and non-structural proteins: identification of an IgG3-restricted recognition site with the envelope 1 protein.

    PubMed Central

    Sällberg, M; Rudén, U; Wahren, B; Magnius, L O

    1993-01-01

    Antibody binding to antigenic regions of hepatitis C virus (HCV) envelope 1 (E1; residues 183-380, E2/non-structural (NS) 1 (residues 380-437), NS1 (residues 643-690), and NS4 (1684-1751) proteins were assayed for 50 sera with antibodies to HCV (anti-HCV) and for 46 sera without anti-HCV. Thirty-four peptides, 18 residues long with an eight-amino acid overlap within each HCV region, were synthesized and tested with all 96 sera. Within the E region 183-380, the major binding site was located to residues 203-220, and was recognized by eight sera. Within the E2/NS1 region 380-437, the peptide covering residues 410-427 was recognized by two sera, and within the NS1 region 643-690, peptides covering residues 663-690 were recognized by four sera. Within the NS4 region 1684-1751, 27 sera were reactive to one or more of the NS4 peptides, and 21 out of these were reactive with peptide 1694-1711. One part of the major binding site could be located to residues 1701-1704, with the sequence Leu-Tyr-Arg-Glu. The IgG1, IgG3 and IgG4 subclasses were reactive with the five antigenic regions of HCV core, residues 1-18, 11-28, 21-38, 51-68 and 101-118. Reactivity to the major envelope site consisted almost exclusively of IgG3, and reactivity to the major site of NS4 consisted only of IgG1. Thus, a non-restricted IgG response to linear HCV-encoded binding sites was found to the core protein, whereas IgG subclass-restricted linear binding sites were found within the E1 protein, and within the NS4 protein. PMID:7680297

  13. Neotropical Bats from Costa Rica harbour Diverse Coronaviruses.

    PubMed

    Moreira-Soto, A; Taylor-Castillo, L; Vargas-Vargas, N; Rodríguez-Herrera, B; Jiménez, C; Corrales-Aguilar, E

    2015-11-01

    Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host-species barrier. For analysing coronavirus diversity in a bat species-rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA-dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus-derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α-CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected. © 2015 Blackwell Verlag GmbH.

  14. Identification and Characterization of a Novel Alpaca Respiratory Coronavirus Most Closely Related to the Human Coronavirus 229E

    PubMed Central

    Crossley, Beate M.; Mock, Richard E.; Callison, Scott A.; Hietala, Sharon K.

    2012-01-01

    In 2007, a novel coronavirus associated with an acute respiratory disease in alpacas (Alpaca Coronavirus, ACoV) was isolated. Full-length genomic sequencing of the ACoV demonstrated the genome to be consistent with other Alphacoronaviruses. A putative additional open-reading frame was identified between the nucleocapsid gene and 3'UTR. The ACoV was genetically most similar to the common human coronavirus (HCoV) 229E with 92.2% nucleotide identity over the entire genome. A comparison of spike gene sequences from ACoV and from HCoV-229E isolates recovered over a span of five decades showed the ACoV to be most similar to viruses isolated in the 1960’s to early 1980’s. The true origin of the ACoV is unknown, however a common ancestor between the ACoV and HCoV-229E appears to have existed prior to the 1960’s, suggesting virus transmission, either as a zoonosis or anthroponosis, has occurred between alpacas and humans. PMID:23235471

  15. Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

    PubMed

    Müller, Christin; Hardt, Martin; Schwudke, Dominik; Neuman, Benjamin W; Pleschka, Stephan; Ziebuhr, John

    2018-02-15

    Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A 2 α (cPLA 2 α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA 2 α activity, which produces lysophospholipids (LPLs) by cleaving at the sn -2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA 2 α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA 2 α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development. IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad

  16. Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

    PubMed

    Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

    2017-10-31

    Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

    PubMed Central

    Nora, Tamara; Bouchonnet, Francine; Labrosse, Béatrice; Charpentier, Charlotte; Mammano, Fabrizio; Clavel, François; Hance, Allan J

    2008-01-01

    Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env) replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1). Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70), and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799), soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5) was also evaluated for a subset of the recombinant viruses (n = 20). Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in characterizing the

  18. Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

    PubMed Central

    Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

    2006-01-01

    The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

  19. Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duquerroy, Stephane; Vigouroux, Armelle; Rottier, Peter J.M.

    2005-05-10

    The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 A) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding 'zippers' that stronglymore » constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 {alpha}-helix.« less

  20. Interferon-β 1a and SARS Coronavirus Replication

    PubMed Central

    Hensley, Lisa E.; Fritz, Elizabeth A.; Karp, Christopher; Huggins, John W.; Geisbert, Thomas W.

    2004-01-01

    A global outbreak of severe acute respiratory syndrome (SARS) caused by a novel coronavirus began in March 2003. The rapid emergence of SARS and the substantial illness and death it caused have made it a critical public health issue. Because no effective treatments are available, an intensive effort is under way to identify and test promising antiviral drugs. Here, we report that recombinant human interferon (IFN)-β 1a potently inhibits SARS coronavirus replication in vitro. PMID:15030704

  1. Role for a Zinc Finger Protein (Zfp111) in Transformation of 208F Rat Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Protein

    PubMed Central

    Hsu, Tom; Phung, An; Choe, Kevin; Kim, Jung Woo

    2015-01-01

    ABSTRACT The native envelope gene (env) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70env) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80env). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus. IMPORTANCE The envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the

  2. Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

    PubMed Central

    Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

    2013-01-01

    A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

  3. Anti-SARS coronavirus agents: a patent review (2008 - present).

    PubMed

    Kumar, Vathan; Jung, Young-Sik; Liang, Po-Huang

    2013-10-01

    A novel coronavirus (CoV), unlike previous typical human coronaviruses (HCoVs), was identified as causative agent for severe acute respiratory syndrome (SARS). SARS first surfaced as a pandemic in late 2002 and originated in southern China. SARS-CoV rapidly spread to > 30 countries by 2003, infecting nearly 8,000 people and causing around 800 fatalities. After 10 years of silence, a 2012 report alarmed researchers about the emergence of a new strain of CoV causing SARS-like disease. To combat SARS, scientists applied for patents on various therapeutic agents, including small-molecule inhibitors targeting the essential proteases, helicase and other proteins of the virus, natural products, approved drugs, molecules binding to the virus, neutralizing antibodies, vaccines, anti-sense RNA, siRNA and ribozyme against SARS-CoV. In this article, the patents published from 2008 to the present for the new therapeutics that could potentially be used in the prophylaxis and treatment of SARS are reviewed. The therapeutic interventions or prophylaxis discussed in this review seems to offer promising solutions to tackle SARS. Rather than being complacent about the results, we should envisage how to transform them into drug candidates that may be useful in combating SARS and related viral infections in the future.

  4. A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

    PubMed

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé; Heidmann, Thierry

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

  5. Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

    PubMed Central

    Webb, Stacy R.; Smith, Stacy E.; Fried, Michael G.

    2018-01-01

    ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral

  6. A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

    PubMed Central

    Ruch, Travis R.; Machamer, Carolyn E.

    2012-01-01

    The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection. PMID:22570613

  7. The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Long; Lok, Shee-Mei; Yu, I-Mei

    2008-09-17

    Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventingmore » fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.« less

  8. Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules.

    PubMed

    Yamada, Tadanori; Jung, Joohee; Seno, Masaharu; Kondo, Akihiko; Ueda, Masakazu; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2012-06-01

    Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

  9. Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

    PubMed Central

    Woo, Patrick C. Y.; Lau, Susanna K. P.; Fan, Rachel Y. Y.; Lau, Candy C. Y.; Wong, Emily Y. M.; Joseph, Sunitha; Tsang, Alan K. L.; Wernery, Renate; Yip, Cyril C. Y.; Tsang, Chi-Ching; Wernery, Ulrich; Yuen, Kwok-Yung

    2016-01-01

    Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1. PMID:27164099

  10. Prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (Acinonyx jubatus).

    PubMed Central

    Heeney, J L; Evermann, J F; McKeirnan, A J; Marker-Kraus, L; Roelke, M E; Bush, M; Wildt, D E; Meltzer, D G; Colly, L; Lukas, J

    1990-01-01

    The extent and progression of exposure to feline infectious peritonitis (FIP) virus in the cheetah, Acinonyx jubatus, was monitored by a world-wide serological survey with indirect fluorescent antibody titers to coronavirus. The indirect fluorescent antibody assay was validated by Western blots, which showed that all indirect fluorescent antibody-positive cheetah sera detected both domestic cat and cheetah coronavirus structural proteins. There was a poor correlation between indirect fluorescent antibody results and the presence of coronaviruslike particles in cheetah feces, suggesting that electron microscopic detection of shed particles may not be an easily interpreted diagnostic parameter for FIP disease. Low, but verifiable (by Western blots [immunoblots]) antibody titers against coronavirus were detected in eight free-ranging cheetahs from east Africa as well as from captive cheetahs throughout the world. Of 20 North American cheetah facilities screened, 9 had cheetahs with measurable antibodies to feline coronavirus. Five facilities showed patterns of an ongoing epizootic. Retrospective FIP virus titers of an FIP outbreak in a cheetah-breeding facility in Oregon were monitored over a 5-year period and are interpreted here in terms of clinical disease progression. During that outbreak the morbidity was over 90% and the mortality was 60%, far greater than any previously reported epizootic of FIP in any cat species. Age of infection was a significant risk factor in this epizootic, with infants (less than 3 months old) displaying significantly higher risk for mortality than subadults or adults. Based upon these observations, empirical generalizations are drawn which address epidemiologic concerns for cheetahs in the context of this lethal infectious agent. Images PMID:2157864

  11. Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

    PubMed Central

    Mielech, Anna M.; Deng, Xufang; Chen, Yafang; Kindler, Eveline; Wheeler, Dorthea L.; Mesecar, Andrew D.; Thiel, Volker; Perlman, Stanley

    2015-01-01

    ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered

  12. Protein composition of the hepatitis A virus quasi-envelope.

    PubMed

    McKnight, Kevin L; Xie, Ling; González-López, Olga; Rivera-Serrano, Efraín E; Chen, Xian; Lemon, Stanley M

    2017-06-20

    The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins ( n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.

  13. Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins.

    PubMed

    Capitanchik, Charlotte; Dixon, Charles; Swanson, Selene K; Florens, Laurence; Kerr, Alastair R W; Schirmer, Eric C

    2018-06-18

    Nuclear envelopathies/laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. One possible explanation of this tissue specificity is that tissue-specific partners become disrupted from larger complexes, but a little investigated alternate hypothesis is that the mutated proteins themselves have tissue-specific splice variants. Here, we analyze RNA-Seq datasets to identify muscle-specific splice variants of nuclear envelope genes that could be relevant to the study of laminopathies, particularly muscular dystrophies, that are not currently annotated in sequence databases. Notably, we found novel isoforms or tissue-specificity of isoforms for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, a regulator of the emerin gene that is linked to Emery-Dreifuss muscular dystrophy. Interestingly, the muscle-specific exon in Lmo7 is rich in serine phosphorylation motifs, suggesting an important regulatory function. Evidence for muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies was also found. Tissue-specific variants were also indicated for several nucleoporins including Nup54, Nup133, Nup153 and Nup358/RanBP2. We confirmed expression of novel Lmo7 and RanBP2 variants with RT-PCR and found that specific knockdown of the Lmo7 variant caused a reduction in myogenic index during mouse C2C12 myogenesis. Global analysis revealed an enrichment of tissue-specific splice variants for nuclear envelope proteins in general compared to the rest of the genome, suggesting that splice variants contribute to regulating its tissue-specific functions.

  14. Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes.

    PubMed

    Petrovsky, Roman; Krohne, Georg; Großhans, Jörg

    2018-03-01

    The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope. Copyright © 2018 Elsevier GmbH. All rights reserved.

  15. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

    PubMed Central

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H.; Züst, Roland; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; Silverman, Robert H.; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C.; Ziebuhr, John; Kalinke, Ulrich

    2017-01-01

    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses. PMID:28158275

  16. Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

    PubMed

    Mullis, Lisa; Saif, Linda J; Zhang, Yongbin; Zhang, Xuming; Azevedo, Marli S P

    2012-05-01

    Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 10⁴ to 1.7 × 10⁶ throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans. Published by Elsevier Ltd.

  17. MERS, SARS and other coronaviruses as causes of pneumonia.

    PubMed

    Yin, Yudong; Wunderink, Richard G

    2018-02-01

    Human coronaviruses (HCoVs) have been considered to be relatively harmless respiratory pathogens in the past. However, after the outbreak of the severe acute respiratory syndrome (SARS) and emergence of the Middle East respiratory syndrome (MERS), HCoVs have received worldwide attention as important pathogens in respiratory tract infection. This review focuses on the epidemiology, pathogenesis and clinical characteristics among SARS-coronaviruses (CoV), MERS-CoV and other HCoV infections. © 2017 Asian Pacific Society of Respirology.

  18. Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

    PubMed

    Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

    2010-12-01

    We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

  19. Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation.

    PubMed

    Kanzawa, Noriyuki; Nishigaki, Kazuo; Hayashi, Takaya; Ishii, Yuichi; Furukawa, Souichi; Niiro, Ayako; Yasui, Fumihiko; Kohara, Michinori; Morita, Kouichi; Matsushima, Kouji; Le, Mai Quynh; Masuda, Takao; Kannagi, Mari

    2006-12-22

    Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-kappaB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.

  20. Nuclear envelope: positioning nuclei and organizing synapses

    PubMed Central

    Razafsky, David; Hodzic, Didier

    2015-01-01

    The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about Atype lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins. PMID:26079712

  1. Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

    PubMed

    Witting, S R; Vallanda, P; Gamble, A L

    2013-10-01

    Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

  2. Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zheng,Q.; Deng, Y.; Liu, J.

    2006-01-01

    Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARSmore » coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an {alpha}-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 {sup o}C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.« less

  3. [Study of neutralization antibodie induced by DNA vaccine of HCV envelope protein 2 in mice].

    PubMed

    Shao, Shengwen; Zhou, Hongchang; Tong, Yimin; Ren, Yanli; Chen, Zhihui

    2011-05-01

    To explore the feasibility of induction of neutralization antibodies against hepatitis C virus (HCV) infection by HCV envelope 2 protein (E2) DNA vaccines immunization. Two kinds of expression plasmids of HCV envelope 2 protein, plasmid pCI-1b661 Delta encoding hydrophobic carboxyl terminal truncated E2 and pCI-1b661 Delta encoding E2 with deletion of hypervariable region 1 (HVR1) and carboxyl terminal, were constructed and respectively transfeted 293T cells, and truncated E2 protein in whole cell lysate and supernatant of 293T cells were analyzed by Western blot. After BALB/c mouse were intramuscularly immunized by the plasmids, sera antibodies against HVR1 were detected by ELISA and the neutralization activity of the antibodies were assayed with HCV pseudotype particle (HCVpp). Both plasmids could express secretary truncated E2 protein. All the mice immunized with plasmid pCI-1b661 produced HVR1 antibodies,while no HVR1 antibodies were detected in pCI-1b661 Delta immunized mice. The sera neutralization percentages against HCVpp in pCI1lb661 Delta and pCI-lb661 Delta immunized mice were (78.5 +/- 13.8)% and (38.7 +/- 6.5)%, respectively (P <0.01). Sera neutralization activity against HCVpp was positive correlated with the level of HVR1 antibodies in pCI-1b661 immunized mice (r = 0.967, P<0.01). DNA vaccines expressing truncated E2 protein could induce neutralization antibodies against HCV, and neutralization antibodies mainly was consisted of the antibodies against HVR1.

  4. Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion.

    PubMed

    Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L

    2015-02-17

    Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.

  5. A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

    PubMed

    Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

    2018-01-01

    The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can

  6. Interaction of SARS and MERS Coronaviruses with the Antiviral Interferon Response.

    PubMed

    Kindler, E; Thiel, V; Weber, F

    2016-01-01

    Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) are the most severe coronavirus (CoV)-associated diseases in humans. The causative agents, SARS-CoV and MERS-CoV, are of zoonotic origin but may be transmitted to humans, causing severe and often fatal respiratory disease in their new host. The two coronaviruses are thought to encode an unusually large number of factors that allow them to thrive and replicate in the presence of efficient host defense mechanisms, especially the antiviral interferon system. Here, we review the recent progress in our understanding of the strategies that highly pathogenic coronaviruses employ to escape, dampen, or block the antiviral interferon response in human cells. © 2016 Elsevier Inc. All rights reserved.

  7. Role of phosphatidylserine receptors in enveloped virus infection.

    PubMed

    Morizono, Kouki; Chen, Irvin S Y

    2014-04-01

    We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear

  8. Evaluation of Serologic and Antigenic Relationships Between Middle Eastern Respiratory Syndrome Coronavirus and Other Coronaviruses to Develop Vaccine Platforms for the Rapid Response to Emerging Coronaviruses

    PubMed Central

    Agnihothram, Sudhakar; Gopal, Robin; Yount, Boyd L.; Donaldson, Eric F.; Menachery, Vineet D.; Graham, Rachel L.; Scobey, Trevor D.; Gralinski, Lisa E.; Denison, Mark R.; Zambon, Maria; Baric, Ralph S.

    2014-01-01

    Background. Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs. Methods. Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs. Results. Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43. Conclusions. Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs. PMID:24253287

  9. N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

    PubMed Central

    Martinet, N; Beninati, S; Nigra, T P; Folk, J E

    1990-01-01

    N1N8-Bis(gamma-glutamyl)spermidine was found in exhaustive proteolytic digests of isolated cell envelopes from human epidermis at levels comparable with those of epsilon-(gamma-glutamyl)lysine. Significantly higher than normal amounts of these compounds, particularly the bis(gamma-glutamyl)polyamine, were observed in envelopes from afflicted areas (scales) of psoriatic patients. These findings support the notions that N1N8-bis(gamma-glutamyl)spermidine, like epsilon-(gamma-glutamyl)lysine, functions in cell envelopes as an enzyme-generated protein cross-link and stabilizing force and that individuals with the chronic, recurrent skin disease, psoriasis, exhibit in involved epidermis abnormal cell-envelope-protein cross-linking. PMID:2241917

  10. Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

    PubMed

    Lu, Jia-hai; Zhang, Ding-mei; Wang, Guo-ling; Guo, Zhong-min; Zhang, Chuan-hai; Tan, Bing-yan; Ouyang, Li-ping; Lin, Li; Liu, Yi-min; Chen, Wei-qing; Ling, Wen-hua; Yu, Xin-bing; Zhong, Nan-shan

    2005-05-05

    The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein. The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The

  11. Fusion of Enveloped Viruses in Endosomes

    PubMed Central

    White, Judith M.; Whittaker, Gary R.

    2016-01-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH, and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion triggering mechanisms. A key take home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors, and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

  12. Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction

    PubMed Central

    Witting, Scott R.; Vallanda, Priya; Gamble, Aisha L.

    2013-01-01

    Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells. PMID:23698741

  13. Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.

    Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17Amore » (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.« less

  14. Detection and characterisation of coronaviruses in migratory and non-migratory Australian wild birds.

    PubMed

    Chamings, Anthony; Nelson, Tiffanie M; Vibin, Jessy; Wille, Michelle; Klaassen, Marcel; Alexandersen, Soren

    2018-04-13

    We evaluated the presence of coronaviruses by PCR in 918 Australian wild bird samples collected during 2016-17. Coronaviruses were detected in 141 samples (15.3%) from species of ducks, shorebirds and herons and from multiple sampling locations. Sequencing of selected positive samples found mainly gammacoronaviruses, but also some deltacoronaviruses. The detection rate of coronaviruses was improved by using multiple PCR assays, as no single assay could detect all coronavirus positive samples. Sequencing of the relatively conserved Orf1 PCR amplicons found that Australian duck gammacoronaviruses were similar to duck gammacoronaviruses around the world. Some sequenced shorebird gammacoronaviruses belonged to Charadriiformes lineages, but others were more closely related to duck gammacoronaviruses. Australian duck and heron deltacoronaviruses belonged to lineages with other duck and heron deltacoronaviruses, but were almost 20% different in nucleotide sequence to other deltacoronavirus sequences available. Deltacoronavirus sequences from shorebirds formed a lineage with a deltacoronavirus from a ruddy turnstone detected in the United States. Given that Australian duck gammacoronaviruses are highly similar to those found in other regions, and Australian ducks rarely come into contact with migratory Palearctic duck species, we hypothesise that migratory shorebirds are the important vector for moving wild bird coronaviruses into and out of Australia.

  15. European Surveillance for Pantropic Canine Coronavirus

    PubMed Central

    Cordonnier, Nathalie; Demeter, Zoltan; Egberink, Herman; Elia, Gabriella; Grellet, Aurélien; Le Poder, Sophie; Mari, Viviana; Martella, Vito; Ntafis, Vasileios; von Reitzenstein, Marcela; Rottier, Peter J.; Rusvai, Miklos; Shields, Shelly; Xylouri, Eftychia; Xu, Zach; Buonavoglia, Canio

    2013-01-01

    Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5′ end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains. PMID:23100349

  16. Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

    PubMed

    Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

    2015-07-01

    Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

  17. Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

    PubMed Central

    Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

    2016-01-01

    ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

  18. Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo‐electron microscopy

    PubMed Central

    Walls, Alexandra; Tortorici, M. Alejandra; Bosch, Berend‐Jan; Frenz, Brandon; Rottier, Peter J. M.; DiMaio, Frank; Rey, Felix A.

    2016-01-01

    Abstract The tremendous pandemic potential of coronaviruses was demonstrated twice in the last 15 years by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor‐binding and membrane fusion functions. Despite their biomedical importance, coronavirus S glycoproteins have proven difficult targets for structural characterization, precluding high‐resolution studies of the biologically relevant trimer. Recent technological developments in single particle cryo‐electron microscopy allowed us to determine the first structure of a coronavirus S glycoprotein trimer which provided a framework to understand the mechanisms of viral entry and suggested potential inhibition strategies for this family of viruses. Here, we describe the key factors that enabled this breakthrough. PMID:27667334

  19. Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

    PubMed

    Li, Ping; Noegel, Angelika A

    2015-11-16

    Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Coronaviruses in guano from Pteropus medius bats in Peradeniya, Sri Lanka.

    PubMed

    Kudagammana, H D W S; Thevanesam, V; Chu, D K W; Eriyagama, N B; Peiris, J S M; Noordeen, F

    2018-03-02

    Bats are a unique group of mammals well suited to be hosts for emerging viruses. With current rates of deforestation and urbanization, redistribution of bat habitats to urban and suburban areas may bring bats into closer contact with livestock and humans. Common flying fox, Pteropus medius (previously known as Pteropus giganteus), forms large communal roosts on treetops, often in close proximity to human habitation in Sri Lanka. This report describes the detection of coronavirus RNA in P. medius bat guano collected in Peradeniya, Sri Lanka. These viruses had >97% nucleotide identity with coronaviruses detected in Cynopterus sphinx, Scotophilus heathii and S. kuhlii bats in Thailand. Pteropus medius is widespread in Asia and appears to excrete group D coronaviruses, which are hitherto confined to bats; however, these findings may have public health implications in the future. © 2018 Blackwell Verlag GmbH.

  1. A New Bat-HKU2–like Coronavirus in Swine, China, 2017

    PubMed Central

    Gong, Lang; Li, Jie; Zhou, Qingfeng; Xu, Zhichao; Chen, Li; Zhang, Yun; Xue, Chunyi

    2017-01-01

    We identified from suckling piglets with diarrhea in China a new bat-HKU2–like porcine coronavirus (porcine enteric alphacoronavirus). The GDS04 strain of this coronavirus shares high aa identities (>90%) with the reported bat-HKU2 strains in Coronaviridae-wide conserved domains, suggesting that the GDS04 strain belongs to the same species as HKU2. PMID:28654418

  2. Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform.

    PubMed

    Mattiacio, Jonelle L; Brewer, Matt; Dewhurst, Stephen

    2017-01-01

    The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform.

  3. Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans

    PubMed Central

    van Boheemen, Sander; de Graaf, Miranda; Lauber, Chris; Bestebroer, Theo M.; Raj, V. Stalin; Zaki, Ali Moh; Osterhaus, Albert D. M. E.; Haagmans, Bart L.; Gorbalenya, Alexander E.; Snijder, Eric J.; Fouchier, Ron A. M.

    2012-01-01

    ABSTRACT A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genome was reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C. PMID:23170002

  4. Recombinant Zika virus envelope protein elicited protective immunity against Zika virus in immunocompetent mice

    PubMed Central

    Liu, Zhihua; Li, Min; Liu, Haitao

    2018-01-01

    Zika virus (ZIKV) has caused great public concerns due to its recent large outbreaks and a close association with microcephaly in fetus and Guillain-Barre syndrome in adults. Rapid development of vaccines against ZIKV is a public health priority. To this end, we have constructed and purified recombinant ZIKV envelope protein using both prokaryotic and eukaryotic expression systems, and then tested their immunogenicity and protective efficacy in immune competent mice. Both protein immunogens elicited humoral and cellular immune responses, and protected immune competent mice from ZIKV challenge in vivo. These products could be further evaluated either as stand-alone vaccine candidate, or used in a prime-and-boost regimen with other forms of ZIKV vaccine. PMID:29590178

  5. Envelope Structures of Gram-Positive Bacteria

    PubMed Central

    Rajagopal, Mithila; Walker, Suzanne

    2016-01-01

    Gram-positive organisms, including the pathogens Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis, have dynamic cell envelopes that mediate interactions with the environment and serve as the first line of defense against toxic molecules. Major components of the cell envelope include peptidoglycan, which is a well-established target for antibiotics, teichoic acids, capsular polysaccharides, surface proteins, and phospholipids. These components can undergo modification to promote pathogenesis, decrease susceptibility to antibiotics and host immune defenses, and enhance survival in hostile environments. This chapter will cover the structure, biosynthesis and important functions of major cell envelope components in Gram-positive bacteria. Possible targets for new antimicrobials will be noted. PMID:26919863

  6. A Signature in HIV-1 Envelope Leader Peptide Associated with Transition from Acute to Chronic Infection Impacts Envelope Processing and Infectivity

    PubMed Central

    Asmal, Mohammed; Hellmann, Ina; Liu, Weimin; Keele, Brandon F.; Perelson, Alan S.; Bhattacharya, Tanmoy; Gnanakaran, S.; Daniels, Marcus; Haynes, Barton F.; Korber, Bette T.; Hahn, Beatrice H.; Shaw, George M.; Letvin, Norman L.

    2011-01-01

    Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission. PMID:21876761

  7. The TIC complex uncovered: The alternative view on the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts.

    PubMed

    Nakai, Masato

    2015-09-01

    Chloroplasts must import thousands of nuclear-encoded preproteins synthesized in the cytosol through two successive protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to fulfill their complex physiological roles. The molecular identity of the TIC translocon had long remained controversial; two proteins, namely Tic20 and Tic110, had been proposed to be central to protein translocation across the inner envelope membrane. Tic40 also had long been considered to be another central player in this process. However, recently, a novel 1-megadalton complex consisting of Tic20, Tic56, Tic100, and Tic214 was identified at the chloroplast inner membrane of Arabidopsis and was demonstrated to constitute a general TIC translocon which functions in concert with the well-characterized TOC translocon. On the other hand, direct interaction between this novel TIC transport system and Tic110 or Tic40 was hardly observed. Consequently, the molecular model for protein translocation across the inner envelope membrane of chloroplasts might need to be extensively revised. In this review article, I intend to propose such alternative view regarding the TIC transport system in contradistinction to the classical view. I also would emphasize importance of reevaluation of previous works in terms of with what methods these classical Tic proteins such as Tic110 or Tic40 were picked up as TIC constituents at the very beginning as well as what actual evidence there were to support their direct and specific involvement in chloroplast protein import. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

    PubMed Central

    Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

  9. GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

    PubMed Central

    Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

    2012-01-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

  10. Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method.

    PubMed

    Takeda, Mitsuhiro; Chang, Chung-ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan-hsiang; Hsu, Yen-lan; Huang, Tai-huang; Kainosho, Masatsune

    2008-07-18

    The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

  11. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection.

    PubMed

    Cheng, Vincent C C; Lau, Susanna K P; Woo, Patrick C Y; Yuen, Kwok Yung

    2007-10-01

    Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.

  12. Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection

    PubMed Central

    Cheng, Vincent C. C.; Lau, Susanna K. P.; Woo, Patrick C. Y.; Yuen, Kwok Yung

    2007-01-01

    Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections. PMID:17934078

  13. Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family.

    PubMed

    Hsueh, Yi-Ching; Flinner, Nadine; Gross, Lucia E; Haarmann, Raimund; Mirus, Oliver; Sommer, Maik S; Schleiff, Enrico

    2017-08-01

    Proteins of the Omp85 family chaperone the membrane insertion of β-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded β-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

  14. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

    PubMed

    Hu, Ben; Zeng, Lei-Ping; Yang, Xing-Lou; Ge, Xing-Yi; Zhang, Wei; Li, Bei; Xie, Jia-Zheng; Shen, Xu-Rui; Zhang, Yun-Zhi; Wang, Ning; Luo, Dong-Sheng; Zheng, Xiao-Shuang; Wang, Mei-Niang; Daszak, Peter; Wang, Lin-Fa; Cui, Jie; Shi, Zheng-Li

    2017-11-01

    A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

  15. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus

    PubMed Central

    Ge, Xing-Yi; Zhang, Wei; Li, Bei; Xie, Jia-Zheng; Shen, Xu-Rui; Zhang, Yun-Zhi; Wang, Ning; Luo, Dong-Sheng; Zheng, Xiao-Shuang; Wang, Mei-Niang; Wang, Lin-Fa

    2017-01-01

    A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases. PMID:29190287

  16. VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

    PubMed

    Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

    2015-04-03

    Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Selection of unadapted, pathogenic SHIVs encoding newly transmitted HIV-1 envelope proteins.

    PubMed

    Del Prete, Gregory Q; Ailers, Braiden; Moldt, Brian; Keele, Brandon F; Estes, Jacob D; Rodriguez, Anthony; Sampias, Marissa; Oswald, Kelli; Fast, Randy; Trubey, Charles M; Chertova, Elena; Smedley, Jeremy; LaBranche, Celia C; Montefiori, David C; Burton, Dennis R; Shaw, George M; Markowitz, Marty; Piatak, Michael; KewalRamani, Vineet N; Bieniasz, Paul D; Lifson, Jeffrey D; Hatziioannou, Theodora

    2014-09-10

    Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

    PubMed

    Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

    2017-07-01

    Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Immunogenicity of Japanese encephalitis virus envelope protein by Hyphantria cunea nuclear polyhedrosis virus vector in guinea pig.

    PubMed

    Lee, Hyung-Hoan; Hong, Seung-Kuk; Yoon, Sang-Ho; Jang, Sung-Jae; Bahk, Young-Yil; Song, Min-Dong; Park, Pyo-Jam; Lee, Kwang-Ho; Kim, Chan-Gil; Kim, Bokyung; Park, Tae-Kyu; Kang, Hyun

    2012-05-01

    Japanese encephalitis virus (JEV) is an important pathogen causing febrile syndrome, encephalitis, and death. Envelop (E) glycoprotein is the major target of inducing neutralizing antibodies and protective immunity in host. In this study, E glycoprotein of JEV was expressed in Spodoptera frugiperd 9 cells as a fusion protein containing a gX signal sequence of pseudorabies virus. This purified HcE recombinant protein was evaluated for their immunogenicity and protective efficacy in guinea pig. The survival rates of guinea pig immunized with HcE protein was significantly increased over that of JE vaccine. This result indicates helpful information for developing a subunit vaccine against JEV.

  20. [Prevalence and clinical characteristics of coronavirus NL63 infection in children hospitalized for acute lower respiratory tract infections in Changsha].

    PubMed

    Zhang, Fei; Zhang, Bing; Xie, Zhi-Ping; Gao, Han-Chun; Zhao, Xin; Zhong, Li-Li; Zhou, Qiong-Hua; Hou, Yun-De; Duan, Zhao-Jun

    2012-04-01

    The main objective of this study was to explore the prevalence and clinical characteristics of human coronavirus NL63 infection in hospitalized children with acute lower respiratory tract infection (ALRTI) in Changsha. Nasopharyngeal aspirates (NPA) samples were collected from 1185 hospitalized children with ALRTI at the People's Hospital of Hunan province, between September 2008 and October 2010. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to screen for coronavirus NL63, which is a 255 bp fragment of a part of N gene. All positive amplification products were confirmed by sequencing and compared with those in GenBank. The overall frequency of coronavirus NL63 infection was 0.8%, 6 (60%) out of the coronavirus NL63 positive patients were detected in summer, 2 in autumn, 1 in spring and winter, respectively. The patients were from 2 months to two and a half years old. The clinical diagnosis was bronchopneumonia (60%), bronchiolitis (30%), and acute laryngotracheal bronchitis (10%). Four of the 10 cases had critical illness, 4 cases had underlying diseases, and 7 cases had mixed infection with other viruses. The homogeneity of coronavirus NL63 with those published in the GenBank at nucleotide levels was 97%-100%. Coronavirus NL63 infection exists in hospitalized children with acute lower respiratory tract infection in Changsha. Coronavirus NL63 infections are common in children under 3 years of age. There is significant difference in the infection rate between the boys and the girls: the boys had higher rate than the girls. The peak of prevalence of the coronavirus NL63 was in summer. A single genetic lineage of coronavirus NL63 was revealed in human subjects in Changsha. Coronavirus NL63 may also be one of the lower respiratory pathogen in China.

  1. Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

    PubMed Central

    2016-01-01

    Rapid progress in disease biomarker discovery has increased the need for robust detection technologies. In the past several years, the designs of many immunoaffinity reagents have focused on lowering costs and improving specificity while also promoting stability. Antibody fragments (scFvs) have long been displayed on the surface of yeast and phage libraries for selection; however, the stable production of such fragments presents challenges that hamper their widespread use in diagnostics. Membrane and cell wall proteins similarly suffer from stability problems when solubilized from their native environment. Recently, cell envelope compositions that maintain membrane proteins in native or native-like lipid environment to improve their stability have been developed. This cell envelope composition approach has now been adapted toward stabilizing antibody fragments by retaining their native cell wall environment. A new class of immunoaffinity reagents has been developed that maintains antibody fragment attachment to yeast cell wall. Herein, we review recent strategies that incorporate cell wall fragments with functional scFvs, which are designed for easy production while maintaining specificity and stability when in use with simple detection platforms. These cell wall based antibody fragments are globular in structure, and heterogeneous in size, with fragments ranging from tens to hundreds of nanometers in size. These fragments appear to retain activity once immobilized onto biosensor surfaces for the specific and sensitive detection of pathogen antigens. They can be quickly and economically generated from a yeast display library and stored lyophilized, at room temperature, for up to a year with little effect on stability. This new format of scFvs provides stability, in a simple and low-cost manner toward the use of scFvs in biosensor applications. The production and “panning” of such antibody cell wall composites are also extremely facile, enabling the rapid

  2. Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro.

    PubMed

    Bacha, Usman; Barrila, Jennifer; Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2004-05-04

    SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain. Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.

  3. Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43

    PubMed Central

    Meessen-Pinard, Mathieu; Le Coupanec, Alain

    2016-01-01

    protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection. PMID:27795420

  4. Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

    PubMed Central

    Reyes-del Valle, Jorge; de la Fuente, Cynthia; Turner, Mallory A.; Springfeld, Christoph; Apte-Sengupta, Swapna; Frenzke, Marie E.; Forest, Amelie; Whidby, Jillian; Marcotrigiano, Joseph; Rice, Charles M.

    2012-01-01

    Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination. PMID:22896607

  5. Inactivation of surrogate coronaviruses on hard surfaces by health care germicides.

    PubMed

    Hulkower, Rachel L; Casanova, Lisa M; Rutala, William A; Weber, David J; Sobsey, Mark D

    2011-06-01

    In the 2003 severe acute respiratory syndrome outbreak, finding viral nucleic acids on hospital surfaces suggested surfaces could play a role in spread in health care environments. Surface disinfection may interrupt transmission, but few data exist on the effectiveness of health care germicides against coronaviruses on surfaces. The efficacy of health care germicides against 2 surrogate coronaviruses, mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV), was tested using the quantitative carrier method on stainless steel surfaces. Germicides were o-phenylphenol/p-tertiary amylphenol) (a phenolic), 70% ethanol, 1:100 sodium hypochlorite, ortho-phthalaldehyde (OPA), instant hand sanitizer (62% ethanol), and hand sanitizing spray (71% ethanol). After 1-minute contact time, for TGEV, there was a log(10) reduction factor of 3.2 for 70% ethanol, 2.0 for phenolic, 2.3 for OPA, 0.35 for 1:100 hypochlorite, 4.0 for 62% ethanol, and 3.5 for 71% ethanol. For MHV, log(10) reduction factors were 3.9 for 70% ethanol, 1.3 for phenolic, 1.7 for OPA, 0.62 for 1:100 hypochlorite, 2.7 for 62% ethanol, and 2.0 for 71% ethanol. Only ethanol reduced infectivity of the 2 coronaviruses by >3-log(10) after 1 minute. Germicides must be chosen carefully to ensure they are effective against viruses such as severe acute respiratory syndrome coronavirus. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  6. Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

    DTIC Science & Technology

    1992-05-12

    hemagglutinating encephalomyelitis virus (HEV), canine coronavirus (CCV), cat FIPV and feline enteric corona virus (FECV), human CVLPs, mouse...While the cat , dog and pig serve as natural hosts for the other coronavirus group 1 viruses , feline infectious peritonitis virus (FIPV), canine...3 2 . Virus Receptors ••••••••.••••••.....•................ 20 3. Viruses Which Cause Common Colds

  7. Construction of recombinant Newcastle disease virus expressing the S1 protein of Turkey enteric coronavirus for use as a bivalent vaccine

    USDA-ARS?s Scientific Manuscript database

    Turkey enteric coronavirus (TCoV) causes a contagious form of enteritis in turkeys, generally recognized in the field by outward signs including diarrhea and decreased weight gain, resulting in severe economic losses for the poultry industry in the US. To date there is no commercial vaccine availab...

  8. SARS-like cluster of circulating bat coronavirus pose threat for human emergence

    PubMed Central

    Menachery, Vineet D.; Yount, Boyd L.; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E.; Plante, Jessica A.; Graham, Rachel L.; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F.; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Shi, Zhengli-Li; Baric, Ralph S.

    2016-01-01

    The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARS-like CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations. PMID:26552008

  9. Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

    PubMed Central

    Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

    2010-01-01

    Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses. PMID:20699222

  10. Fluorescence molecular painting of enveloped viruses.

    PubMed

    Metzner, Christoph; Kochan, Feliks; Dangerfield, John A

    2013-01-01

    In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment. Results from the FMP-flow cytometry approach were verified by immunoblotting and enzyme-linked immunosorbent assay. Since the modification strategy is applicable to a broad range of proteins and viruses, variations of this method may be useful in a range of research and applied applications from bio-distribution studies to vaccine development and targeted infection for gene delivery.

  11. A mouse model for MERS coronavirus-induced acute respiratory distress syndrome.

    PubMed

    Cockrell, Adam S; Yount, Boyd L; Scobey, Trevor; Jensen, Kara; Douglas, Madeline; Beall, Anne; Tang, Xian-Chun; Marasco, Wayne A; Heise, Mark T; Baric, Ralph S

    2016-11-28

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR-Cas9 gene editing to modify the mouse genome to encode two amino acids (positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS, including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-induced ARDS.

  12. The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination.

    PubMed

    Hu, Yong; Li, Wei; Gao, Ting; Cui, Yan; Jin, Yanwen; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2017-04-15

    Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein. IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response. Copyright © 2017 American Society for Microbiology.

  13. The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination

    PubMed Central

    Hu, Yong; Li, Wei; Gao, Ting; Cui, Yan; Jin, Yanwen; Li, Ping; Ma, Qingjun

    2017-01-01

    ABSTRACT Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein. IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response. PMID:28148787

  14. Cloning and expression of an envelope gene of West Nile virus and evaluation of the protein for use in an IgM ELISA.

    PubMed

    Saxena, Divyasha; Parida, Manmohan; Rao, Putcha Venkata L; Kumar, Jyoti S

    2013-04-01

    West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and epidemiologic control in areas where multiple flaviviruses are endemic. The coexistence of WNV along with other members of flaviviruses like dengue and Japanese encephalitis in India has complicated the serodiagnosis due to cross-reactive antigens. In the present study, the development and evaluation of a highly sensitive and specific IgM enzyme-linked immunosorbent assay (ELISA) using the recombinant envelope protein (rWNV-Env) for rapid, early, and accurate diagnosis of WNV are reported. The gene coding for the envelope protein of WNV was cloned and expressed in pET 28a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rWNV-Env protein was optimized having no cross reactivity with healthy human serum. Furthermore, the specificity of this assay was confirmed by cross checking with serum samples obtained from patients with dengue and Japanese encephalitis viruses. The comparative evaluation of this rWNV-Env protein-specific IgM ELISA with plaque reduction neutralization test assay using 105 acute phase of clinical samples revealed 95% concordance with sensitivity and specificity of 92% and 97%, respectively. The positive and negative predictive values of recombinant-based Env ELISA were 94% and 96%, respectively. The recombinant envelope protein-based WNV-specific ELISA reported in this study will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

    PubMed

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

    2016-11-01

    Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether

  16. Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope.

    PubMed

    Siegel, Sara D; Reardon, Melissa E; Ton-That, Hung

    2017-01-01

    In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

  17. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.

    PubMed

    Scobey, Trevor; Yount, Boyd L; Sims, Amy C; Donaldson, Eric F; Agnihothram, Sudhakar S; Menachery, Vineet D; Graham, Rachel L; Swanstrom, Jesica; Bove, Peter F; Kim, Jeeho D; Grego, Sonia; Randell, Scott H; Baric, Ralph S

    2013-10-01

    Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

  18. The Tegument Protein UL71 of Human Cytomegalovirus Is Involved in Late Envelopment and Affects Multivesicular Bodies ▿

    PubMed Central

    Schauflinger, Martin; Fischer, Daniela; Schreiber, Andreas; Chevillotte, Meike; Walther, Paul; Mertens, Thomas; von Einem, Jens

    2011-01-01

    Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV. PMID:21289123

  19. A Novel Chemical Compound for Inhibition of SARS Coronavirus Helicase.

    PubMed

    Lee, Jin-Moo; Cho, Jin-Beom; Ahn, Hee-Chul; Jung, Woong; Jeong, Yong-Joo

    2017-11-28

    We have discovered a novel chemical compound, (E)-3-(furan-2-yl)- N -(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC 50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

  20. Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

    PubMed

    Han, Mee-Jung; Lee, Sang Yup; Hong, Soon Ho

    2012-04-01

    Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

  1. Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3.

    PubMed

    Wong, Hui Hui; Fung, To Sing; Fang, Shouguo; Huang, Mei; Le, My Tra; Liu, Ding Xiang

    2018-02-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age

    PubMed Central

    Wernery, Ulrich; Rasoul, IHassab El; Wong, Emily YM; Joseph, Marina; Chen, Yixin; Jose, Shanty; Tsang, Alan KL; Patteril, Nissy Annie Georgy; Chen, Honglin; Elizabeth, Shyna K; Yuen, Kwok-Yung; Joseph, Sunitha; Xia, Ningshao; Wernery, Renate; Lau, Susanna KP; Woo, Patrick CY

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted. PMID:26632876

  3. A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age.

    PubMed

    Wernery, Ulrich; El Rasoul, I Hassab; Wong, Emily Y M; Joseph, Marina; Chen, Yixin; Jose, Shanty; Tsang, Alan K L; Patteril, Nissy Annie Georgy; Chen, Honglin; Elizabeth, Shyna K; Yuen, Kwok-Yung; Joseph, Sunitha; Xia, Ningshao; Wernery, Renate; Lau, Susanna K P; Woo, Patrick C Y

    2015-12-02

    Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted.Emerging Microbes & Infections (2015) 4, e74; doi:10.1038/emi.2015.74; published online 2 December 2015.

  4. SARS-associated Coronavirus Transmitted from Human to Pig

    PubMed Central

    Chen, Weijun; Yan, Minghua; Yang, Ling; Ding, Boliang; He, Bo; Wang, Yingzhen; Liu, Xiuli; Liu, Chenhui; Zhu, Hui; You, Bo; Huang, Shengyong; Zhang, Jiangguo; Mu, Feng; Xiang, Zhao; Feng, Xiaoli; Wen, Jie; Fang, Jianqiu; Yu, Jun; Yang, Huanming

    2005-01-01

    Severe acute respiratory syndrome–associated coronavirus (SARS-CoV) was isolated from a pig during a survey for possible routes of viral transmission after a SARS epidemic. Sequence and epidemiology analyses suggested that the pig was infected by a SARS-CoV of human origin. PMID:15757562

  5. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

  6. Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

    PubMed Central

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-01-01

    Our knowledge of the binding sites for neutralizing antibodies (NAbs) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B-cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

  7. The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

    PubMed

    Xiang, Xingwei; Chen, Lin; Guo, Aiqin; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

    2011-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

    PubMed

    Bedgood, R M; Stallcup, M R

    1992-04-05

    The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

  9. Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli

    PubMed Central

    Matsumoto, Ayaka; Sakamoto, Chiyomi; Matsumori, Haruka; Katahira, Jun; Yasuda, Yoko; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Goldberg, Ilya G; Matsuura, Nariaki; Nakao, Mitsuyoshi; Saitoh, Noriko; Hieda, Miki

    2016-01-01

    ABSTRACT A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells. PMID:26962703

  10. Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli.

    PubMed

    Matsumoto, Ayaka; Sakamoto, Chiyomi; Matsumori, Haruka; Katahira, Jun; Yasuda, Yoko; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Goldberg, Ilya G; Matsuura, Nariaki; Nakao, Mitsuyoshi; Saitoh, Noriko; Hieda, Miki

    2016-01-01

    A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells.

  11. Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection

    PubMed Central

    Totura, Allison L.; Whitmore, Alan; Agnihothram, Sudhakar; Schäfer, Alexandra; Katze, Michael G.; Heise, Mark T.

    2015-01-01

    ABSTRACT Toll-like receptors (TLRs) are sensors that recognize molecular patterns from viruses, bacteria, and fungi to initiate innate immune responses to invading pathogens. The emergence of highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) is a concern for global public health, as there is a lack of efficacious vaccine platforms and antiviral therapeutic strategies. Previously, it was shown that MyD88, an adaptor protein necessary for signaling by multiple TLRs, is a required component of the innate immune response to mouse-adapted SARS-CoV infection in vivo. Here, we demonstrate that TLR3−/−, TLR4−/−, and TRAM−/− mice are more susceptible to SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung function, increased lung pathology, and higher viral titers. Distinct alterations in inflammation were present in TRIF−/− mice infected with SARS-CoV, including excess infiltration of neutrophils and inflammatory cell types that correlate with increased pathology of other known causes of acute respiratory distress syndrome (ARDS), including influenza virus infections. Aberrant proinflammatory cytokine, chemokine, and interferon-stimulated gene (ISG) signaling programs were also noted following infection of TRIF−/− mice that were similar to those seen in human patients with poor disease outcome following SARS-CoV or MERS-CoV infection. These findings highlight the importance of TLR adaptor signaling in generating a balanced protective innate immune response to highly pathogenic coronavirus infections. PMID:26015500

  12. HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

    PubMed

    Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

    2018-03-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

  13. Physiological temperatures reduce dimerization of dengue and Zika virus recombinant envelope proteins.

    PubMed

    Kudlacek, Stephan T; Premkumar, Lakshmanane; Metz, Stefan W; Tripathy, Ashutosh; Bobkov, Andrey A; Payne, Alexander Matthew; Graham, Stephen; Brackbill, James A; Miley, Michael J; de Silva, Aravinda M; Kuhlman, Brian

    2018-06-08

    The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 μm at 25 °C to 50 μm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine. © 2018 Kudlacek et al.

  14. Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; del Carmen Parquet, Maria; Morita, Kouichi

    2005-01-01

    Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ121 protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ121 protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ121 protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ121 protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist. PMID:16002634

  15. Mechanism of Dissolution of Envelopes of the Extreme Halophile Halobacterium cutirubrum1

    PubMed Central

    Onishi, H.; Kushner, D. J.

    1966-01-01

    Onishi, H. (National Research Council, Ottawa, Ontario, Canada), and D. J. Kushner. Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum. J. Bacteriol. 91:646–652. 1966.—Envelopes of Halobacterium cutirubrum dissolved rapidly in media of low ionic strength. Heating partially inhibited breakdown, probably because of nonspecific protein coagulation rather than inactivation of a lytic enzyme(s). Dissolution of envelopes in water did not involve splitting of peptide bonds or protein-lipid bonds, or any extensive breakdown of carbohydrate polymers. Dissolution was increased by alcohols and urea, even at high salt concentrations, but was not affected by metabolic inhibitors. Thus, no evidence was found for a dilution-activated lytic enzyme that contributes to envelope breakdown. Cells of H. cutirubrum were stable in 2 m NaCl, but lysis occurred in 2 m KCl or NH4Cl. This lysis did not involve an extensive breakdown of the envelope. No evidence for different sites of Na+, K+, and NH4+ action was obtained from the pattern of release of envelope constituents in different concentrations of these salts. Ultracentrifugation studies showed that adding salts to envelopes that had been dissolved in water led to a nonspecific reaggregation of envelope material. No difference was seen between the effects of KCl and NaCl, except at 3 to 4 m concentrations where KCl caused more aggregation. The preferential effect of Na+ on intact cells is probably due to its ability specifically to prevent leakage rather than to an overall effect on envelope integrity. Images PMID:5883109

  16. Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

    PubMed

    Benedicto, Ignacio; Molina-Jiménez, Francisca; Barreiro, Olga; Maldonado-Rodríguez, Alejandra; Prieto, Jesús; Moreno-Otero, Ricardo; Aldabe, Rafael; López-Cabrera, Manuel; Majano, Pedro L

    2008-10-01

    Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

  17. Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains.

    PubMed

    Yuan, Yuan; Cao, Duanfang; Zhang, Yanfang; Ma, Jun; Qi, Jianxun; Wang, Qihui; Lu, Guangwen; Wu, Ying; Yan, Jinghua; Shi, Yi; Zhang, Xinzheng; Gao, George F

    2017-04-10

    The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD). We captured two states of the RBD with receptor binding region either buried (lying state) or exposed (standing state), demonstrating an inherently flexible RBD readily recognized by the receptor. Further sequence conservation analysis of six human-infecting coronaviruses revealed that the fusion peptide, HR1 region and the central helix are potential targets for eliciting broadly neutralizing antibodies.

  18. Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production

    PubMed Central

    Chouljenko, Dmitry V.; Jambunathan, Nithya; Chouljenko, Vladimir N.; Naderi, Misagh; Brylinski, Michal; Caskey, John R.

    2016-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both

  19. Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.

    PubMed

    Gross, C H; Russell, R L; Rohrmann, G F

    1994-05-01

    To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.

  20. Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1.

    PubMed

    Lu, Shuai; Wang, Yanqun; Chen, Yingzhu; Wu, Bingjie; Qin, Kun; Zhao, Jincun; Lou, Yongliang; Tan, Wenjie

    2017-06-02

    Although canine respiratory coronavirus (CRCoV) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of CRCoV (South Korea strain K37) has been obtained to date. Genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. Herein, we report a unique CRCoV strain, denoted strain BJ232, derived from a CRCoV-positive dog with a mild respiratory infection. Phylogenetic analysis based on complete genome of all available coronaviruses consistently show that CRCoV BJ232 is most closely related to human coronavirus OC43 (HCoV-OC43) and BCoV, forming a separate clade that split off early from other Betacoronavirus 1. Based on the phylogenetic and SimPlot analysis we propose that CRCoV-K37 was derived from genetic recombination between CRCoV-BJ232 and BCoV. In detail, spike (S) gene of CRCoV-K37 clustered with CRCoV-BJ232. However orf1ab, membrane (M) and nucleocapsid (N) genes were more related to Bovine coronavirus (BCoV) than CRCoV-B232. Molecular epidemic analysis confirmed the prevalence of CRCoV-BJ232 lineage around the world for a long time. Recombinant events among Betacoronavirus 1 may have implications for CRCoV transmissibility. All these findings provide further information regarding the origin of CRCoV. Copyright © 2017. Published by Elsevier B.V.

  1. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    PubMed

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Serial femtosecond X-ray diffraction of enveloped virus microcrystals

    DOE PAGES

    Lawrence, Robert M.; Conrad, Chelsie E.; Zatsepin, Nadia A.; ...

    2015-08-20

    Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ~700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ~40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is a pertinent step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

  3. Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells

    PubMed Central

    Tilton, Susan C.; Menachery, Vineet D.; Gralinski, Lisa E.; Schäfer, Alexandra; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo M.; Chang, Jean; Luna, Maria L.; Long, Casey E.; Shukla, Anil K.; Bankhead, Armand R.; Burkett, Susan E.; Zornetzer, Gregory; Tseng, Chien-Te Kent; Metz, Thomas O.; Pickles, Raymond; McWeeney, Shannon; Smith, Richard D.; Katze, Michael G.; Waters, Katrina M.; Baric, Ralph S.

    2013-01-01

    The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo. PMID:23365422

  4. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Young, Kevin G.; University of Ottawa Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages precedingmore » the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.« less

  5. Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Johnson, Chassidy; Jahid, Sohail; Voelker, Dennis R.

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods ofmore » time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.« less

  6. Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

    PubMed

    Békés, Miklós; van der Heden van Noort, Gerbrand J; Ekkebus, Reggy; Ovaa, Huib; Huang, Tony T; Lima, Christopher D

    2016-05-19

    Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Nuclear envelopathies: a complex LINC between nuclear envelope and pathology.

    PubMed

    Janin, Alexandre; Bauer, Delphine; Ratti, Francesca; Millat, Gilles; Méjat, Alexandre

    2017-08-30

    Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of "nuclear envelopathies" is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

  8. Structure-based drug design for envelope protein E2 uncovers a new class of bovine viral diarrhea inhibitors that block virus entry.

    PubMed

    Pascual, María José; Merwaiss, Fernando; Leal, Emilse; Quintana, María Eugenia; Capozzo, Alejandra V; Cavasotto, Claudio N; Bollini, Mariela; Alvarez, Diego E

    2018-01-01

    Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC 50 of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV. Copyright © 2017 Elsevier B.V. All rights

  9. Discovery of novel bovine viral diarrhea inhibitors using structure-based virtual screening on the envelope protein E2

    NASA Astrophysics Data System (ADS)

    Bollini, Mariela; Leal, Emilse S.; Adler, Natalia S.; Aucar, María G.; Fernández, Gabriela A.; Pascual, María J.; Merwaiss, Fernando; Alvarez, Diego E.; Cavasotto, Claudio N.

    2018-03-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. BVDV causes both acute and persistent infections in cattle, leading to substantial financial losses to the livestock industry each year. The global prevalence of persistent BVDV infection and the lack of a highly effective antiviral therapy have spurred intensive efforts to discover and develop novel anti-BVDV therapies in the pharmaceutical industry. Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. We performed prospective small-molecule high-throughput docking to identify molecules that likely bind to the region delimited by domains I and II of the envelope protein E2 of BVDV. Several structurally different compounds were purchased or synthesized, and assayed for antiviral activity against BVDV. Five of the selected compounds were active displaying IC50 values in the low- to mid-micromolar range. For these compounds, their possible binding determinants were characterized by molecular dynamics simulations. A common pattern of interactions between active molecules and aminoacid residues in the binding site in E2 was observed. These findings could offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals.

  10. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein.

    PubMed

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-06-15

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

  11. Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

    PubMed Central

    Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

    2004-01-01

    A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

  12. Coronavirus antibody detection in cats by computer-assisted kinetics-based enzyme-linked immunosorbent assay (KELA): field studies.

    PubMed

    Barlough, J E; Jacobson, R H; Sorresso, G P; Lynch, T J; Scott, F W

    1986-07-01

    A total of 2238 feline serum samples submitted to the New York State Diagnostic Laboratory over a 1-year period were tested for the presence of coronavirus antibodies, using the computer-assisted, kinetics-based enzyme-linked immunosorbent assay (KELA). Cats from which sera were obtained were categorized by sex, age, breed, and disease status, and variations in mean antibody titers for different sub-classifications within each category were analyzed by computerized statistical analysis. As expected, higher mean antibody titers were recorded for cats with feline infectious peritonitis, and for cats with a recent history of possible coronavirus exposure. However, an unexpected inverse relationship between coronavirus antibody titer and age was also found. Certain cattery-oriented pure breeds appeared to have higher mean antibody titers, because their sample populations contained a higher percentage of younger cats and cats of unknown age-groups which, over-all, had higher mean titers. Taken together, the data substantiated the efficacy of the computer-assisted KELA for routine detection of serum coronavirus antibodies in cats.

  13. HERV Envelope Proteins: Physiological Role and Pathogenic Potential in Cancer and Autoimmunity

    PubMed Central

    Grandi, Nicole; Tramontano, Enzo

    2018-01-01

    Human endogenous retroviruses (HERVs) are relics of ancient infections accounting for about the 8% of our genome. Despite their persistence in human DNA led to the accumulation of mutations, HERVs are still contributing to the human transcriptome, and a growing number of findings suggests that their expression products may have a role in various diseases. Among HERV products, the envelope proteins (Env) are currently highly investigated for their pathogenic properties, which could likely be participating to several disorders with complex etiology, particularly in the contexts of autoimmunity and cancer. In fact, HERV Env proteins have been shown, on the one side, to trigger both innate and adaptive immunity, prompting inflammatory, cytotoxic and apoptotic reactions; and, on the other side, to prevent the immune response activation, presenting immunosuppressive properties and acting as immune downregulators. In addition, HERV Env proteins have been shown to induce abnormal cell-cell fusion, possibly contributing to tumor development and metastasizing processes. Remarkably, even highly defective HERV env genes and alternative env splicing variants can provide further mechanisms of pathogenesis. A well-known example is the HERV-K(HML2) env gene that, depending on the presence or the absence of a 292-bp deletion, can originate two proteins of different length (Np9 and Rec) proposed to have oncogenic properties. The understanding of their involvement in complex pathological disorders made HERV Env proteins potential targets for therapeutic interventions. Of note, a monoclonal antibody directed against a HERV-W Env is currently under clinical trial as therapeutic approach for multiple sclerosis, representing the first HERV-based treatment. The present review will focus on the current knowledge of the HERV Env expression, summarizing its role in human physiology and its possible pathogenic effects in various cancer and autoimmune disorders. It moreover analyzes HERV Env

  14. Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice.

    PubMed

    Gralinski, Lisa E; Menachery, Vineet D; Morgan, Andrew P; Totura, Allison L; Beall, Anne; Kocher, Jacob; Plante, Jessica; Harrison-Shostak, D Corinne; Schäfer, Alexandra; Pardo-Manuel de Villena, Fernando; Ferris, Martin T; Baric, Ralph S

    2017-06-07

    Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1-58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2 , an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2 -/- mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses. Copyright © 2017 Gralinski et al.

  15. Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice

    PubMed Central

    Gralinski, Lisa E.; Menachery, Vineet D.; Morgan, Andrew P.; Totura, Allison L.; Beall, Anne; Kocher, Jacob; Plante, Jessica; Harrison-Shostak, D. Corinne; Schäfer, Alexandra; Pardo-Manuel de Villena, Fernando; Ferris, Martin T.; Baric, Ralph S.

    2017-01-01

    Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1–58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2−/− mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses. PMID:28592648

  16. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

    PubMed Central

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

    2016-01-01

    ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

  17. Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya.

    PubMed

    Kiyuka, Patience K; Agoti, Charles N; Munywoki, Patrick K; Njeru, Regina; Bett, Anne; Otieno, James R; Otieno, Grieven P; Kamau, Everlyn; Clark, Taane G; van der Hoek, Lia; Kellam, Paul; Nokes, D James; Cotten, Matthew

    2018-05-05

    Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.

  18. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.

    PubMed

    Menachery, Vineet D; Yount, Boyd L; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E; Plante, Jessica A; Graham, Rachel L; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F; Randell, Scott H; Lanzavecchia, Antonio; Marasco, Wayne A; Shi, Zhengli-Li; Baric, Ralph S

    2015-12-01

    The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.

  19. Global patterns in coronavirus diversity

    PubMed Central

    Johnson, Christine K.; Greig, Denise J.; Kramer, Sarah; Che, Xiaoyu; Wells, Heather; Hicks, Allison L.; Joly, Damien O.; Wolfe, Nathan D.; Daszak, Peter; Karesh, William; Lipkin, W. I.; Morse, Stephen S.; Mazet, Jonna A. K.

    2017-01-01

    Abstract Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the ‘pre-emergent’ diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence. PMID:28630747

  20. The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

    PubMed Central

    Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

    2009-01-01

    Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved

  1. Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay.

    PubMed

    Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; Del Carmen Parquet, Maria; Morita, Kouichi

    2005-07-01

    Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.

  2. Cerebral pyogranuloma associated with systemic coronavirus infection in a ferret.

    PubMed

    Gnirs, K; Quinton, J F; Dally, C; Nicolier, A; Ruel, Y

    2016-01-01

    A 2-year-old male ferret was presented with central nervous system signs. Computed tomography (CT) of the brain revealed a well-defined contrast-enhancing lesion on the rostral forebrain that appeared extraparenchymal. Surgical excision of the mass was performed and the ferret was euthanised during the procedure. Histopathology of the excised mass showed multiple meningeal nodular lesions with infiltrates of epithelioid macrophages, occasionally centred on degenerated neutrophils and surrounded by a broad rim of plasma cells, features consistent with pyogranulomatous meningitis. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis. Definitive diagnosis was assessed by immunohistochemistry, confirming a ferret systemic coronavirus (FSCV) associated disease. This is the first case of coronavirus granuloma described on CT-scan in the central nervous system of a ferret. © 2015 British Small Animal Veterinary Association.

  3. Targeted entry of enveloped viruses: measles and herpes simplex virus I.

    PubMed

    Navaratnarajah, Chanakha K; Miest, Tanner S; Carfi, Andrea; Cattaneo, Roberto

    2012-02-01

    We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus (HSV) is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and HSV can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Arabidopsis WPP-Domain Proteins Are Developmentally Associated with the Nuclear Envelope and Promote Cell DivisionW⃞

    PubMed Central

    Patel, Shalaka; Rose, Annkatrin; Meulia, Tea; Dixit, Ram; Cyr, Richard J.; Meier, Iris

    2004-01-01

    The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference–based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity. PMID:15548735

  5. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

    PubMed

    Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

    2006-07-01

    Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.

  6. Role of HIV-2 envelope in Lv2-mediated restriction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reuter, Sandra; Kaumanns, Patrick; Buschhorn, Sabine B.

    2005-02-05

    We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1more » (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-A{delta}env-GFP, ROD-A{delta}env-RFP, or ROD-A{delta}env-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E)« less

  7. UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

    PubMed Central

    Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

    2001-01-01

    The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus

  8. Three dimensional model of severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain and molecular design of severe acute respiratory syndrome coronavirus helicase inhibitors

    NASA Astrophysics Data System (ADS)

    Hoffmann, Marcin; Eitner, Krystian; von Grotthuss, Marcin; Rychlewski, Leszek; Banachowicz, Ewa; Grabarkiewicz, Tomasz; Szkoda, Tomasz; Kolinski, Andrzej

    2006-05-01

    The modeling of the severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain was performed using the protein structure prediction Meta Server and the 3D Jury method for model selection, which resulted in the identification of 1JPR, 1UAA and 1W36 PDB structures as suitable templates for creating a full atom 3D model. This model was further utilized to design small molecules that are expected to block an ATPase catalytic pocket thus inhibit the enzymatic activity. Binding sites for various functional groups were identified in a series of molecular dynamics calculation. Their positions in the catalytic pocket were used as constraints in the Cambridge structural database search for molecules having the pharmacophores that interacted most strongly with the enzyme in a desired position. The subsequent MD simulations followed by calculations of binding energies of the designed molecules were compared to ATP identifying the most successful candidates, for likely inhibitors—molecules possessing two phosphonic acid moieties at distal ends of the molecule.

  9. Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: insights into mechanisms of general viral fusion and inhibitor design.

    PubMed

    Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

    2014-05-01

    Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation=0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. © 2014 The Protein Society.

  10. Genomic and serological detection of bat coronavirus from bats in the Philippines.

    PubMed

    Tsuda, Shumpei; Watanabe, Shumpei; Masangkay, Joseph S; Mizutani, Tetsuya; Alviola, Phillip; Ueda, Naoya; Iha, Koichiro; Taniguchi, Satoshi; Fujii, Hikaru; Kato, Kentaro; Horimoto, Taisuke; Kyuwa, Shigeru; Yoshikawa, Yasuhiro; Akashi, Hiroomi

    2012-12-01

    Bat coronavirus (BtCoV) is assumed to be a progenitor of severe acute respiratory syndrome (SARS)-related coronaviruses. To explore the distribution of BtCoVs in the Philippines, we collected 179 bats and detected viral RNA from intestinal or fecal samples by RT-PCR. The overall prevalence of BtCoVs among bats was 29.6 %. Phylogenetic analysis of the partial RNA-dependent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others belonged to the genus Betacoronavirus. Western blotting revealed that 66.5 % of bat sera had antibodies to BtCoV. These surveys suggested the endemic presence of BtCoVs in the Philippines.

  11. Single-round selection yields a unique retroviral envelope utilizing GPR172A as its host receptor.

    PubMed

    Mazari, Peter M; Linder-Basso, Daniela; Sarangi, Anindita; Chang, Yehchung; Roth, Monica J

    2009-04-07

    The recognition by a viral envelope of its cognate host-cell receptor is the initial critical step in defining the viral host-range and tissue specificity. This study combines a single-round of selection of a random envelope library with a parallel cDNA screen for receptor function to identify a distinct retroviral envelope/receptor pair. The 11-aa targeting domain of the modified feline leukemia virus envelope consists of a constrained peptide. Critical to the binding of the constrained peptide envelope to its cellular receptor are a pair of internal cysteines and an essential Trp required for maintenance of titers >10(5) lacZ staining units per milliliter. The receptor used for viral entry is the human GPR172A protein, a G-protein-coupled receptor isolated from osteosarcoma cells. The ability to generate unique envelopes capable of using tissue- or disease-specific receptors marks an advance in the development of efficient gene-therapy vectors.

  12. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    PubMed

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  13. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

    PubMed Central

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Müller, Marcel A.; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-01-01

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

  14. The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation

    PubMed Central

    Ding, Zhen; Fang, Liurong; Yuan, Shuangling; Zhao, Ling; Wang, Xunlei; Long, Siwen; Wang, Mohan; Wang, Dang; Foda, Mohamed Frahat; Xiao, Shaobo

    2017-01-01

    Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-β production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N–PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-β production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins. PMID:28591694

  15. The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

    PubMed

    Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

    2003-02-11

    A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

  16. The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

    PubMed Central

    Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

    2003-01-01

    A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE. PMID:12694627

  17. Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.

    ABSTRACT Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impactmore » on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting. IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2'O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN

  18. Enhanced production and immunological characterization of recombinant West Nile virus envelope domain III protein.

    PubMed

    Tripathi, Nagesh K; Karothia, Divyanshi; Shrivastava, Ambuj; Banger, Swati; Kumar, Jyoti S

    2018-05-13

    West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation. Wet biomass obtained after batch and fed-batch cultivation processes was 11.2 g and 84 g/L of culture respectively. Protein yield after affinity purification was 5.76 mg and 5.81 mg/g wet cell weight after batch and fed-batch processes respectively. The purified WNV EDIII elicited specific antibodies in rabbits, confirming its immunogenicity. Moreover, the antibodies were able to neutralize WNV in vitro. These results established that the refolded and purified WNV EDIII could be a potential vaccine candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

    PubMed Central

    Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai

    2015-01-01

    Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses. PMID:26019210

  20. Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein.

    PubMed

    Wang, Yue-Dan; Chen, Wei Feng

    2004-11-01

    To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.

  1. Cleavage and Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Human Airway Trypsin-Like Protease ▿

    PubMed Central

    Bertram, Stephanie; Glowacka, Ilona; Müller, Marcel A.; Lavender, Hayley; Gnirss, Kerstin; Nehlmeier, Inga; Niemeyer, Daniela; He, Yuxian; Simmons, Graham; Drosten, Christian; Soilleux, Elizabeth J.; Jahn, Olaf; Steffen, Imke; Pöhlmann, Stefan

    2011-01-01

    The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients. PMID:21994442

  2. Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

    PubMed

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.

  3. Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein

    USGS Publications Warehouse

    Fassbinder-Orth, C. A.; Hofmeister, Erik K.; Weeks-Levy, C.; Karasov, W.H.

    2009-01-01

    West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 ??g or 100 ??g/dose), of E protein intramuscularly (IM, 20 ??g/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds. ?? 2009 American Association of Avian Pathologists.

  4. Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

    PubMed

    Corman, V M; Eckerle, I; Bleicker, T; Zaki, A; Landt, O; Eschbach-Bludau, M; van Boheemen, S; Gopal, R; Ballhause, M; Bestebroer, T M; Muth, D; Müller, M A; Drexler, J F; Zambon, M; Osterhaus, A D; Fouchier, R M; Drosten, C

    2012-09-27

    We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5–6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

  5. Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

    PubMed Central

    McAuley, Alexander J.; Torres, Maricela; Plante, Jessica A.; Huang, Claire Y.-H.; Bente, Dennis A.

    2016-01-01

    ABSTRACT Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, with in vivo pathogenesis often not being correlated with in vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric

  6. Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

    PubMed

    McAuley, Alexander J; Torres, Maricela; Plante, Jessica A; Huang, Claire Y-H; Bente, Dennis A; Beasley, David W C

    2016-05-01

    Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a

  7. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

    PubMed Central

    Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

    2006-01-01

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

  8. Incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus.

    PubMed

    Nishiura, H; Mizumoto, K; Ejima, K; Zhong, Y; Cowling, Bj; Omori, R

    2012-10-18

    Non-specific symptoms of acute respiratory viral infections make it difficult for many countries without ongoing transmission of a novel coronavirus to rule out other possibilities including influenza before isolating imported febrile individuals with a possible exposure history. The incubation period helps differential diagnosis, and up to two days is suggestive of influenza. It is worth including the incubation period in the case definition of novel coronavirus infection.

  9. Association of seropositivity for influenza and coronaviruses with history of mood disorders and suicide attempts.

    PubMed

    Okusaga, Olaoluwa; Yolken, Robert H; Langenberg, Patricia; Lapidus, Manana; Arling, Timothy A; Dickerson, Faith B; Scrandis, Debra A; Severance, Emily; Cabassa, Johanna A; Balis, Theodora; Postolache, Teodor T

    2011-04-01

    Anecdotal reports of mood disorder following infection with common respiratory viruses with neurotropic potential have been in existence since the last century. Nevertheless, systematic studies on the association between these viruses and mood disorders are lacking. Influenza A, B and coronavirus antibody titers were measured in 257 subjects with recurrent unipolar and bipolar disorder and healthy controls, by SCID. Pearson's χ² tests and logistic regression models were used to analyze associations between seropositivity for coronaviruses, influenza A and B viruses and the following: a) history of recurrent mood disorders b) having attempted suicide in the past c) uni- vs. bi-polarity and d) presence of psychotic symptoms during mood episodes. Seropositivity for influenza A (p=0.004), B (p<0.0001) and coronaviruses (p<0.0001) were associated with history of mood disorders but not with the specific diagnosis of unipolar or bipolar depression. Seropositivity for influenza B was significantly associated with a history of suicide attempt (p=0.001) and history of psychotic symptoms (p=0.005). The design was cross-sectional. Socioeconomic factors, inflammatory markers, and axis II psychopathology were not assessed. The association of seropositivity for influenza and coronaviruses with a history of mood disorders, and influenza B with suicidal behavior require replication in larger longitudinal samples. The need for these studies is additionally supported by the high incidence of these viral infections, the high prevalence of mood disorders, and resilience of suicide epidemics. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections.

    PubMed

    Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan; Veesler, David

    2017-11-01

    Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S 2 ' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity. IMPORTANCE Coronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is

  11. Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway

    PubMed Central

    2012-01-01

    Background The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here. Findings Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs). Conclusions These findings add TBC1D20 to the network of host factors regulating HIV replication cycle. PMID:22260459

  12. The Metabolite Transporters of the Plastid Envelope: An Update

    PubMed Central

    Facchinelli, Fabio; Weber, Andreas P. M.

    2011-01-01

    The engulfment of a photoautotrophic cyanobacterium by a primitive mitochondria-bearing eukaryote traces back to more than 1.2 billion years ago. This single endosymbiotic event not only provided the early petroalgae with the metabolic capacity to perform oxygenic photosynthesis, but also introduced a plethora of other metabolic routes ranging from fatty acids and amino acids biosynthesis, nitrogen and sulfur assimilation to secondary compounds synthesis. This implicated the integration and coordination of the newly acquired metabolic entity with the host metabolism. The interface between the host cytosol and the plastidic stroma became of crucial importance in sorting precursors and products between the plastid and other cellular compartments. The plastid envelope membranes fulfill different tasks: they perform important metabolic functions, as they are involved in the synthesis of carotenoids, chlorophylls, and galactolipids. In addition, since most genes of cyanobacterial origin have been transferred to the nucleus, plastidial proteins encoded by nuclear genes are post-translationally transported across the envelopes through the TIC–TOC import machinery. Most importantly, chloroplasts supply the photoautotrophic cell with photosynthates in form of reduced carbon. The innermost bilayer of the plastidic envelope represents the permeability barrier for the metabolites involved in the carbon cycle and is literally stuffed with transporter proteins facilitating their transfer. The intracellular metabolite transporters consist of polytopic proteins containing membrane spans usually in the number of four or more α-helices. Phylogenetic analyses revealed that connecting the plastid with the host metabolism was mainly a process driven by the host cell. In Arabidopsis, 58% of the metabolite transporters are of host origin, whereas only 12% are attributable to the cyanobacterial endosymbiont. This review focuses on the metabolite transporters of the inner envelope

  13. Expression, Purification, Crystallization of Two Major Envelope Proteins from White Spot Syndrome Virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang,X.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapor-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 Mmore » sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 {angstrom} resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 {angstrom}. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 {angstrom}, and diffracts to 2.0 {angstrom} resolution.« less

  14. Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

    PubMed

    Melloy, Patricia G; Rose, Mark D

    2017-09-15

    Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction.

    PubMed

    Artier, Juliana; da Silva Zandonadi, Flávia; de Souza Carvalho, Flávia Maria; Pauletti, Bianca Alves; Leme, Adriana Franco Paes; Carnielli, Carolina Moretto; Selistre-de-Araujo, Heloisa Sobreiro; Bertolini, Maria Célia; Ferro, Jesus Aparecido; Belasque Júnior, José; de Oliveira, Julio Cezar Franco; Novo-Mansur, Maria Teresa Marques

    2018-01-01

    Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  16. Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

    PubMed Central

    Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

    2014-01-01

    Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. PMID:24519901

  17. The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

    PubMed

    Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L

    2016-01-01

    FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

  18. Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture

    PubMed Central

    Ding, Zhao-Ying; Huang, Yu-Cheng; Lee, Myong-Chol; Tseng, Min-Jen; Chi, Ya-Hui

    2017-01-01

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture. PMID:28716842

  19. Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies ▿

    PubMed Central

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F.; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections. PMID:21084464

  20. Genetically Diverse Coronaviruses in Wild Bird Populations of Northern England

    PubMed Central

    Savage, Carol; Naylor, Clive; Bennett, Malcolm; Chantrey, Julian; Jones, Richard

    2009-01-01

    Infectious bronchitis virus (IBV) causes a costly respiratory viral disease of chickens. The role of wild birds in the epidemiology of IBV is poorly understood. We detected diverse coronaviruses by PCR in wildfowl and wading birds in England. Sequence analysis showed some viruses to be related to IBV. PMID:19624927

  1. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

    PubMed

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

  2. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

    PubMed Central

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  3. A soluble envelope protein of endogenous retrovirus (FeLIX) present in serum of domestic cats mediates infection of a pathogenic variant of feline leukemia virus.

    PubMed

    Sakaguchi, Shoichi; Shojima, Takayuki; Fukui, Daisuke; Miyazawa, Takayuki

    2015-03-01

    T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion defective because in its PHQ motif, a gammaretroviral consensus motif in the N terminus of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and is expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in the blood and that FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats and not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus. © 2015 The Authors.

  4. Severe Acute Respiratory Syndrome–associated Coronavirus Infection

    PubMed Central

    Ip, Margaret; Ng, KC; Wu, Alan; Lee, Nelson; Rainer, Timothy H.; Joynt, Gavin M.; Sung, Joseph J. Y.; Tam, John S.

    2003-01-01

    Whether severe acute respiratory syndrome–associated coronavirus (SARS-CoV) infection can be asymptomatic is unclear. We examined the seroprevalence of SARS-CoV among 674 healthcare workers from a hospital in which a SARS outbreak had occurred. A total of 353 (52%) experienced mild self-limiting illnesses, and 321 (48%) were asymptomatic throughout the course of these observations. None of these healthcare workers had antibody to SARS CoV, indicating that subclinical or mild infection attributable to SARS CoV in adults is rare. PMID:14718090

  5. Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

    PubMed Central

    Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

    2013-01-01

    Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

  6. Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

    USDA-ARS?s Scientific Manuscript database

    The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

  7. Automated analysis of cell migration and nuclear envelope rupture in confined environments.

    PubMed

    Elacqua, Joshua J; McGregor, Alexandra L; Lammerding, Jan

    2018-01-01

    Recent in vitro and in vivo studies have highlighted the importance of the cell nucleus in governing migration through confined environments. Microfluidic devices that mimic the narrow interstitial spaces of tissues have emerged as important tools to study cellular dynamics during confined migration, including the consequences of nuclear deformation and nuclear envelope rupture. However, while image acquisition can be automated on motorized microscopes, the analysis of the corresponding time-lapse sequences for nuclear transit through the pores and events such as nuclear envelope rupture currently requires manual analysis. In addition to being highly time-consuming, such manual analysis is susceptible to person-to-person variability. Studies that compare large numbers of cell types and conditions therefore require automated image analysis to achieve sufficiently high throughput. Here, we present an automated image analysis program to register microfluidic constrictions and perform image segmentation to detect individual cell nuclei. The MATLAB program tracks nuclear migration over time and records constriction-transit events, transit times, transit success rates, and nuclear envelope rupture. Such automation reduces the time required to analyze migration experiments from weeks to hours, and removes the variability that arises from different human analysts. Comparison with manual analysis confirmed that both constriction transit and nuclear envelope rupture were detected correctly and reliably, and the automated analysis results closely matched a manual analysis gold standard. Applying the program to specific biological examples, we demonstrate its ability to detect differences in nuclear transit time between cells with different levels of the nuclear envelope proteins lamin A/C, which govern nuclear deformability, and to detect an increase in nuclear envelope rupture duration in cells in which CHMP7, a protein involved in nuclear envelope repair, had been depleted

  8. Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

    PubMed Central

    Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

    1998-01-01

    Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

  9. A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

    PubMed

    Wu, Kailang; Chen, Lang; Peng, Guiqing; Zhou, Wenbo; Pennell, Christopher A; Mansky, Louis M; Geraghty, Robert J; Li, Fang

    2011-06-01

    How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

  10. Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

    PubMed

    Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

    2016-03-01

    Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides.

  11. Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

    PubMed

    Kelly, Amélie A; Kalisch, Barbara; Hölzl, Georg; Schulze, Sandra; Thiele, Juliane; Melzer, Michael; Roston, Rebecca L; Benning, Christoph; Dörmann, Peter

    2016-09-20

    Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

  12. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

    PubMed Central

    Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

  13. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    PubMed

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  14. T-Cell response profiling to biological threat agents including the SARS coronavirus.

    PubMed

    Gioia, C; Horejsh, D; Agrati, C; Martini, F; Capobianchi, M R; Ippolito, G; Poccia, F

    2005-01-01

    The emergence of pathogens such as SARS and the increased threat of bioterrorism has stimulated the development of novel diagnostic assays for differential diagnosis. Rather than focusing on the detection of an individual pathogen component, we have developed a T cell profiling system to monitor responses to the pathogens in an array format. Using a matrix of antigens specific for different pathogens, a specific T cell profile was generated for each individual by monitoring the intracellular production of interferon-gamma by flow cytometry. This assay allows for the testing of multiple proteins or peptides at a single time and provides a quantitative and phenotypic assessment of CD4(+) and CD8(+) responding cells. We present profiling examples for several positive individuals, including those vaccinated with the smallpox and anthrax vaccines. We also show antigen optimization for the SARS-hCoV, as studies revealed that these proteins contain peptides which cross-react with more common coronaviruses, a cause of the common cold. The T cell array is an early and sensitive multiplex measure of active infection, exposure to a pathogen, or effective, recent vaccination.

  15. Clinico-epidemiological characteristics of acute respiratory infections caused by coronavirus OC43, NL63 and 229E.

    PubMed

    Reina, J; López-Causapé, C; Rojo-Molinero, E; Rubio, R

    2014-12-01

    Acute respiratory infection is a very common condition in the general population. The majority of these infections are due to viruses. This study attempted to determine the clinical and epidemiological characteristics of adult patients with respiratory infection by the coronavirus OC43, NL63 and 229E. Between January 2013 and February 2014, we prospectively studied all patients with suspected clinical respiratory infection by taking throat swabs and performing a reverse transcription polymerase chain reaction in search of coronavirus. In 48 cases (7.0% of the 686 enrolled patients; 12.6% of the 381 in whom a virus was detected) the presence of a coronavirus demonstrated. In 24 cases, the virus was OC43 (50%); in 14 cases, the virus was NL63 (29%); and in 10 cases, the virus was 229E (21%). The mean age was 54.5 years, with a slight predominance of men. The most common clinical presentations were nonspecific influenza symptoms (43.7%), pneumonia (29.2%) and chronic obstructive pulmonary disease exacerbation (8.3%). Fifty-two percent of the patients required hospitalization, and 2 patients required intensive care. There were no deaths. Acute respiratory infections caused by coronavirus mainly affect middle-aged male smokers, who are often affected by previous diseases. The most common clinical picture has been nonspecific influenza symptoms. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  16. Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation.

    PubMed

    Conley, Lynn; Tao, Yinying; Henry, Alexis; Koepf, Edward; Cecchini, Douglas; Pieracci, John; Ghose, Sanchayita

    2017-04-01

    Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings

  17. Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

    PubMed

    Fukushi, Shuetsu; Fukuma, Aiko; Kurosu, Takeshi; Watanabe, Shumpei; Shimojima, Masayuki; Shirato, Kazuya; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ohnishi, Kazuo; Ato, Manabu; Melaku, Simenew Keskes; Sentsui, Hiroshi; Saijo, Masayuki

    2018-01-01

    Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Enveloping Aerodynamic Decelerator

    NASA Technical Reports Server (NTRS)

    Nock, Kerry T. (Inventor); Aaron, Kim M. (Inventor); McRonald, Angus D. (Inventor); Gates, Kristin L. (Inventor)

    2018-01-01

    An inflatable aerodynamic deceleration method and system is provided for use with an atmospheric entry payload. The inflatable aerodynamic decelerator includes an inflatable envelope and an inflatant, wherein the inflatant is configured to fill the inflatable envelope to an inflated state such that the inflatable envelope surrounds the atmospheric entry payload, causing aerodynamic forces to decelerate the atmospheric entry payload.

  19. Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope1[OPEN

    PubMed Central

    Tanabe, Noriaki; Clarke, Adrian K.

    2016-01-01

    The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import. PMID:26586836

  20. Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus.

    PubMed

    Josset, Laurence; Menachery, Vineet D; Gralinski, Lisa E; Agnihothram, Sudhakar; Sova, Pavel; Carter, Victoria S; Yount, Boyd L; Graham, Rachel L; Baric, Ralph S; Katze, Michael G

    2013-04-30

    A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS-CoV. Using a human lung epithelial cell line and global transcriptomic profiling, we identified differences in the host response between HCoV-EMC and SARS-CoV. This enables rapid assessment of viral properties and the

  1. Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

    PubMed

    Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

    2013-08-01

    A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  2. The nuclear envelope from basic biology to therapy.

    PubMed

    Worman, Howard J; Foisner, Roland

    2010-02-01

    The nuclear envelope has long been a focus of basic research for a highly specialized group of cell biologists. More recently, an expanding group of scientists and physicians have developed a keen interest in the nuclear envelope since mutations in the genes encoding lamins and associated proteins have been shown to cause a diverse range of human diseases often called laminopathies or nuclear envelopathies. Most of these diseases have tissue-selective phenotypes, suggesting that the nuclear envelope must function in cell-type- and developmental-stage-specific processes such as chromatin organization, regulation of gene expression, controlled nucleocytoplasmic transport and response to stress in metazoans. On 22-23 April 2009, Professor Christopher Hutchison organized the 4th British Nuclear Envelope Disease and Chromatin Organization meeting at the College of St Hild and St Bede at Durham University, sponsored by the Biochemical Society. In attendance were investigators with one common interest, the nuclear envelope, but with diverse expertise and training in animal and plant cell biology, genetics, developmental biology and medicine. We were each honoured to be keynote speakers. This issue of Biochemical Society Transactions contains papers written by some of the presenters at this scientifically exciting meeting, held in a bucolic setting where the food was tasty and the wine flowed freely. Perhaps at the end of this excellent meeting more questions were raised than answered, which will stimulate future research. However, what became clear is that the nuclear envelope is a cellular structure with critical functions in addition to its traditional role as a barrier separating the nuclear and cytoplasmic compartments in interphase eukaryotic cells.

  3. Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin.

    PubMed

    Ren, Wuze; Qu, Xiuxia; Li, Wendong; Han, Zhenggang; Yu, Meng; Zhou, Peng; Zhang, Shu-Yi; Wang, Lin-Fa; Deng, Hongkui; Shi, Zhengli

    2008-02-01

    Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV), which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs (SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organizations and high sequence identities, with the main exception of the N terminus of the spike protein (S), known to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in structure and in function. The significance of these findings in relation to virus origin, virus recombination, and host switching is discussed.

  4. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, bothmore » sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.« less

  5. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

    PubMed

    Bower, Joseph F; Green, Thomas D; Ross, Ted M

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

  6. Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication.

    PubMed

    Sakai, Yusuke; Kawachi, Kengo; Terada, Yutaka; Omori, Hiroko; Matsuura, Yoshiharu; Kamitani, Wataru

    2017-10-01

    Infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mRNA occur. Although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. We demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. In addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. Finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cDNA clone and replicon system of SARS-CoV. These findings provide clues to the mechanism of the replication/transcription complex assembly of SARS-CoV and could reveal an antiviral target for the treatment of betacoronavirus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Dynamic assembly of brambleberry mediates nuclear envelope fusion during early development.

    PubMed

    Abrams, Elliott W; Zhang, Hong; Marlow, Florence L; Kapp, Lee; Lu, Sumei; Mullins, Mary C

    2012-08-03

    To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. The occlusion-derived virus envelope protein ODV-E56 is required for optimal oral infectivity but is not essential for virus binding and fusion

    USDA-ARS?s Scientific Manuscript database

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine the role of ODV-E56 in oral infectivity, we produced recombinant EGFP-expressing AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lac...

  9. Nasal Colonisation by Staphylococcus aureus Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

    PubMed Central

    Mulcahy, Michelle E.; Geoghegan, Joan A.; Monk, Ian R.; O'Keeffe, Kate M.; Walsh, Evelyn J.

    2012-01-01

    Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB+ S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB− mutant colonised wild-type mice less efficiently than the parental ClfB+ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB− mutant in the Lor−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal

  10. The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McDermott, Jason E.; Mitchell, Hugh D.; Gralinski, Lisa E.

    The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ antiimmune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identifymore » genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), promote pathogenesis through a parallel feed-forward circuit that promotes inflammation. These results are consistent with previous studies showing the role of over-stimulation of the inflammatory response to SARS-CoV in pathogenesis. We conclude that the critical balance between immune response and inflammation can be manipulated to improve the outcome of the infection. Further, our study provides two potential therapeutic strategies for mitigating the effects of SARS-CoV infection, and may provide insight into treatment strategies for Middle East Respiratory Syndrome Coronavirus (MERS-CoV).« less

  11. Multi-scale signed envelope inversion

    NASA Astrophysics Data System (ADS)

    Chen, Guo-Xin; Wu, Ru-Shan; Wang, Yu-Qing; Chen, Sheng-Chang

    2018-06-01

    Envelope inversion based on modulation signal mode was proposed to reconstruct large-scale structures of underground media. In order to solve the shortcomings of conventional envelope inversion, multi-scale envelope inversion was proposed using new envelope Fréchet derivative and multi-scale inversion strategy to invert strong contrast models. In multi-scale envelope inversion, amplitude demodulation was used to extract the low frequency information from envelope data. However, only to use amplitude demodulation method will cause the loss of wavefield polarity information, thus increasing the possibility of inversion to obtain multiple solutions. In this paper we proposed a new demodulation method which can contain both the amplitude and polarity information of the envelope data. Then we introduced this demodulation method into multi-scale envelope inversion, and proposed a new misfit functional: multi-scale signed envelope inversion. In the numerical tests, we applied the new inversion method to the salt layer model and SEG/EAGE 2-D Salt model using low-cut source (frequency components below 4 Hz were truncated). The results of numerical test demonstrated the effectiveness of this method.

  12. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins.

    PubMed

    Maric, Martina; Haugo, Alison C; Dauer, William; Johnson, David; Roller, Richard J

    2014-07-01

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Human Immunodeficiency Virus Envelope Protein Gp120 Induces Proliferation but Not Apoptosis in Osteoblasts at Physiologic Concentrations

    PubMed Central

    Cummins, Nathan W.; Klicpera, Anna; Sainski, Amy M.; Bren, Gary D.; Khosla, Sundeep; Westendorf, Jennifer J.; Badley, Andrew D.

    2011-01-01

    Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism. PMID:21931863

  14. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Venkatagopalan, Pavithra; School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401; Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteinemore » tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.« less

  15. Antiviral Peptides Targeting the West Nile Virus Envelope Protein▿

    PubMed Central

    Bai, Fengwei; Town, Terrence; Pradhan, Deepti; Cox, Jonathan; Ashish; Ledizet, Michel; Anderson, John F.; Flavell, Richard A.; Krueger, Joanna K.; Koski, Raymond A.; Fikrig, Erol

    2007-01-01

    West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses. PMID:17151121

  16. Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: Exploring the proteosurfaceome of Escherichia coli using sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin.

    PubMed

    Monteiro, Ricardo; Chafsey, Ingrid; Leroy, Sabine; Chambon, Christophe; Hébraud, Michel; Livrelli, Valérie; Pizza, Mariagrazia; Pezzicoli, Alfredo; Desvaux, Mickaël

    2018-06-15

    Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the sulfo-NHS-SS-biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight sulfo-NHS-SS-biotin. Actually, the sulfo-NHS-SS-biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. While generally pass over in silence, some periplasmic proteins

  17. Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus

    PubMed Central

    Josset, Laurence; Menachery, Vineet D.; Gralinski, Lisa E.; Agnihothram, Sudhakar; Sova, Pavel; Carter, Victoria S.; Yount, Boyd L.; Graham, Rachel L.; Baric, Ralph S.; Katze, Michael G.

    2013-01-01

    ABSTRACT A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. PMID:23631916

  18. Cellular phosphoinositides and the maturation of bluetongue virus, a non-enveloped capsid virus

    PubMed Central

    2013-01-01

    Background Bluetongue virus (BTV), a member of Orbivirus genus in the Reoviridae family is a double capsid virus enclosing a genome of 10 double-stranded RNA segments. A non-structural protein of BTV, NS3, which is associated with cellular membranes and interacts with outer capsid proteins, has been shown to be involved in virus morphogenesis in infected cells. In addition, studies have also shown that during the later stages of virus infection NS3 behaves similarly to HIV protein Gag, an enveloped viral protein. Since Gag protein is known to interact with membrane lipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and one of the known binding partners of NS3, cellular protein p11 also interacts with annexin a PI(4,5)P2 interacting protein, this study was designed to understand the role of this negatively charged membrane lipid in BTV assembly and maturation. Methods Over expression of cellular enzymes that either depleted cells of PI(4,5)P2 or altered the distribution of PI(4,5)P2, were used to analyze the effect of the lipid on BTV maturation at different times post-infection. The production of mature virus particles was monitored by plaque assay. Microscopic techniques such as confocal microscopy and electron microscopy (EM) were also undertaken to study localization of virus proteins and virus particles in cells, respectively. Results Initially, confocal microscopic analysis demonstrated that PI(4,5)P2 not only co-localized with NS3, but it also co-localized with VP5, one of the outer capsid proteins of BTV. Subsequently, experiments involving depletion of cellular PI(4,5)P2 or its relocation demonstrated an inhibitory effect on normal BTV maturation and it also led to a redistribution of BTV proteins within the cell. The data was supported further by EM visualization showing that modulation of PI(4,5)P2 in cells indeed resulted in less particle production. Conclusion This study to our knowledge, is the first report demonstrating involvement of PI(4,5)P2

  19. Bovine coronavirus (BCV) infections in transported commingled beef cattle and sole-source ranch calves

    PubMed Central

    Fulton, Robert W.; Step, Douglas L.; Wahrmund, Jackie; Burge, Lurinda J.; Payton, Mark E.; Cook, Billy J.; Burken, Dirk; Richards, Chris J.; Confer, Anthony W.

    2011-01-01

    This study investigated bovine coronavirus (BCV) in both beef calves direct from the ranch and commingled, mixed-source calves obtained from an auction market. The level of BCV-neutralizing antibodies found in the calves varied among ranches in 2 different studies in a retained-ownership program (ROP), from the ranch to the feedlot. Calves with low levels of BCV-neutralizing antibodies (16 or less) were more likely to be treated for bovine respiratory disease (BRD) than those with higher titers. In 3 studies of commingled, mixed-source calves, BCV was recovered from calves at entry to the feedlot and the infections were cleared by day 8. The BCV was identified in lung samples [bronchoalveolar lavage (BAL) collection] as well as in nasal swabs. Calves with low levels of BCV-neutralizing antibodies at entry were most likely to be shedding BCV. Bovine coronavirus was isolated from both healthy and sick calves, but not from sick calves after 4 d arrival at the feedlot. Bovine coronavirus (BCV) should be considered along with other bovine respiratory viruses in the diagnosis of etiologies in bovine respiratory disease, especially for animals that become sick shortly after arrival. If approved vaccines are developed, it would be best to carry out vaccination programs before calves are weaned, giving them sufficient time to gain active immunity before commingling with other cattle. PMID:22210995

  20. Middle East Respiratory Syndrome Coronavirus Antibodies in Dromedary Camels, Bangladesh, 2015

    PubMed Central

    Islam, Ariful; Rostal, Melinda K.; Islam, Shariful; Rahman, Mohammed Ziaur; Hossain, Mohammed Enayet; Uzzaman, Mohammed Salim; Munster, Vincent J.; Peiris, Malik; Flora, Meerjady Sabrina; Rahman, Mahmudur; Daszak, Peter

    2018-01-01

    Dromedary camels are bred domestically and imported into Bangladesh. In 2015, of 55 camels tested for Middle East respiratory syndrome coronavirus in Dhaka, 17 (31%) were seropositive, including 1 bred locally. None were PCR positive. The potential for infected camels in urban markets could have public health implications and warrants further investigation. PMID:29664373

  1. Serologic follow-up of Middle East Respiratory Syndrome Coronavirus Cases and Contacts - Abu Dhabi, United Arab Emirates.

    PubMed

    Al Hosani, Farida Ismail; Kim, Lindsay; Khudhair, Ahmed; Pham, Huong; Al Mulla, Mariam; Al Bandar, Zyad; Pradeep, Krishna; Elkheir, Kheir Abou; Weber, Stefan; Khoury, Mary; Donnelly, George; Younis, Naima; El Saleh, Feda; Abdalla, Muna; Imambaccus, Hala; Haynes, Lia M; Thornburg, Natalie J; Harcourt, Jennifer L; Miao, Congrong; Tamin, Azaibi; Hall, Aron J; Russell, Elizabeth S; Harris, Aaron M; Kiebler, Craig; Mir, Roger A; Pringle, Kimberly; Alami, Negar N; Abedi, Glen R; Gerber, Susan I

    2018-06-13

    Although there is evidence of person-to-person transmission of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent that disease severity affects transmission. A sero-epidemiological investigation was conducted among Middle East Respiratory Syndrome Coronavirus (MERS-CoV) case-patients and their household contacts to investigate transmission risk in Abu Dhabi, United Arab Emirates. Cases diagnosed between January 1, 2013-May 9, 2014 and their household contacts were approached for enrollment. Demographic, clinical, and exposure history data were collected. Sera were screened by MERS-CoV nucleocapsid protein (N) ELISA and indirect immunofluorescence, with results confirmed by microneutralization assay. Ninety-one percent (n=31/34) of case-patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. MERS-CoV antibodies were detected in 13 of 24 (54%) cases with available sera, including 3 asymptomatic, 9 mildly symptomatic, and 1 severely symptomatic case-patient. No serologic evidence of MERS-CoV transmission was found among 105 household contacts with available sera. Transmission of MERS-CoV was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. These results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission.

  2. Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

    2013-01-01

    The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

  3. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Claire Y.-H., E-mail: CHuang1@cdc.go; Butrapet, Siritorn; Moss, Kelly J.

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could notmore » re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.« less

  4. Mutation analysis of the fusion domain region of St. Louis encephalitis virus envelope protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trainor, Nicole B.; Crill, Wayne D.; Roberson, Jill A.

    2007-04-10

    The immune response to flavivirus infections produces both species-specific and flavivirus cross-reactive antibodies. The presence of cross-reactive antibodies complicates serodiagnosis of flavivirus infections, especially secondary infections caused by a heterologous virus. A successful public health response to the growing global threat posed by flaviviruses necessitates the development of virus-specific diagnostic antigens. The flavivirus envelope (E) glycoprotein is the principle antigen stimulating protective immunity during infection. Using recombinant St. Louis encephalitis virus-like particles (VLPs), we have identified amino acid residues involved in flavivirus cross-reactive epitope determinants. Most significant among the residues studied are three highly conserved amino acids in the fusionmore » peptide: Gly104, Gly106, and Leu107. Substitutions of these residues dramatically influenced VLP secretion and cross-reactive monoclonal antibody reactivity. These results provide critical insight into the antigenic structure of the flaviviral E protein and toward development of species-specific diagnostic antigens that should improve both flavivirus diagnosis and estimates of disease burden.« less

  5. Envelope-receptor interactions in Nipah virus pathobiology.

    PubMed

    Lee, Benhur

    2007-04-01

    Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope-receptor interactions in NiV pathobiology. Finally, we also describe how galectin-1 (an innate immune defense lectin) can interact with specific N-glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens.

  6. A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

    PubMed

    Troupin, Andrea; Londono-Renteria, Berlin; Conway, Michael J; Cloherty, Erin; Jameson, Samuel; Higgs, Stephen; Vanlandingham, Dana L; Fikrig, Erol; Colpitts, Tonya M

    2016-09-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Searching for animal models and potential target species for emerging pathogens: Experience gained from Middle East respiratory syndrome (MERS) coronavirus.

    PubMed

    Vergara-Alert, Júlia; Vidal, Enric; Bensaid, Albert; Segalés, Joaquim

    2017-06-01

    Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013-2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV), which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV), associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed.

  8. Rapid detection of MERS coronavirus-like viruses in bats: pote1ntial for tracking MERS coronavirus transmission and animal origin.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Chen, Yixin; Wong, Emily Y M; Chan, Kwok-Hung; Chen, Honglin; Zhang, Libiao; Xia, Ningshao; Yuen, Kwok-Yung

    2018-03-07

    Recently, we developed a monoclonal antibody-based rapid nucleocapsid protein detection assay for diagnosis of MERS coronavirus (MERS-CoV) in humans and dromedary camels. In this study, we examined the usefulness of this assay to detect other lineage C betacoronaviruses closely related to MERS-CoV in bats. The rapid MERS-CoV nucleocapsid protein detection assay was tested positive in 24 (88.9%) of 27 Tylonycteris bat CoV HKU4 (Ty-BatCoV-HKU4) RNA-positive alimentary samples of Tylonycteris pachypus and 4 (19.0%) of 21 Pipistrellus bat CoV HKU5 (Pi-BatCoV-HKU5) RNA-positive alimentary samples of Pipistrellus abramus. There was significantly more Ty-BatCoV-HKU4 RNA-positive alimentary samples than Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive by the rapid MERS-CoV nucleocapsid protein detection assay (P < 0.001 by Chi-square test). The rapid assay was tested negative in all 51 alimentary samples RNA-positive for alphacoronaviruses (Rhinolophus bat CoV HKU2, Myotis bat CoV HKU6, Miniopterus bat CoV HKU8 and Hipposideros batCoV HKU10) and 32 alimentary samples positive for lineage B (SARS-related Rhinolophus bat CoV HKU3) and lineage D (Rousettus bat CoV HKU9) betacoronaviruses. No significant difference was observed between the viral loads of Ty-BatCoV-HKU4/Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive and negative by the rapid test (Mann-Witney U test). The rapid MERS-CoV nucleocapsid protein detection assay is able to rapidly detect lineage C betacoronaviruses in bats. It detected significantly more Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5 because MERS-CoV is more closely related to Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5. This assay will facilitate rapid on-site mass screening of animal samples for ancestors of MERS-CoV and tracking transmission in the related bat species.

  9. The use of specific antibodies to mediate fusion between Sendai virus envelopes and living cells.

    PubMed

    Loyter, A; Tomasi, M; Gitman, A G; Etinger, L; Nussbaum, O

    1984-01-01

    Incubation of Sendai virus particles with non-ionic detergents such as Triton X-100 completely solubilizes the viral envelopes. Removal of the detergent from the supernatant (which contains the two main viral glycoproteins) leads to the formation of fusogenic, reconstituted viral envelopes. Soluble macromolecules such as DNA or proteins can be enclosed within the reconstituted vesicles, while membrane components can be inserted into the viral envelopes. Fusion of such loaded or 'hybrid' reconstituted envelopes with living cells in culture results in either microinjection or transfer of the viral components to the recipient cells. Thus such reconstituted envelopes can serve as efficient carriers for the introduction of macromolecules of biological interest into living cells in culture. A more specific vehicle has been constructed by chemically coupling anti-cell membrane antibodies (anti-human erythrocyte antibody) to the viral envelope. Such antibody-bearing intact virus particles or reconstituted envelopes bound to and fused with virus receptor-depleted cells. In addition, anti-Sendai virus antibodies were coupled to neuraminidase-treated human erythrocytes. Such antibodies mediated the binding and fusion of intact Sendai virus particles and their reconstituted envelopes to virus receptor-depleted cells.

  10. Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms.

    PubMed

    Xie, Qian; Cao, Yujuan; Su, Juan; Wu, Jie; Wu, Xianbo; Wan, Chengsong; He, Mingliang; Ke, Changwen; Zhang, Bao; Zhao, Wei

    2017-08-01

    Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.

  11. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    PubMed Central

    2011-01-01

    Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family) and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E) is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE) containing the envelope gene (env) of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine. PMID:21619598

  12. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera.

    PubMed

    Barros, Maria C E S; Galasso, Tatiane G C M; Chaib, Antônio J M; Degallier, Nicolas; Nagata, Tatsuya; Ribeiro, Bergmann M

    2011-05-27

    Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family) and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E) is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE) containing the envelope gene (env) of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine.

  13. Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

    PubMed

    Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

    2016-08-01

    Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties.

    PubMed

    Bukin, Yu S; Dzhioev, Yu P; Tkachev, S E; Kozlova, I V; Paramonov, A I; Ruzek, D; Qu, Z; Zlobin, V I

    2017-06-15

    This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Enteric disease in postweaned beef calves associated with a Bovine coronavirus clade 2

    USDA-ARS?s Scientific Manuscript database

    Bovine coronavirus (BoCV) infections are associated with varied clinical presentations including neonatal diarrhea, winter dysentery in dairy cattle, and respiratory disease in various ages of cattle. This report presents information on BoCV infections associated with enteric disease of postweaned b...

  16. Immunogenicity of NYVAC Prime-Protein Boost Human Immunodeficiency Virus Type 1 Envelope Vaccination and Simian-Human Immunodeficiency Virus Challenge of Nonhuman Primates.

    PubMed

    Saunders, Kevin O; Santra, Sampa; Parks, Robert; Yates, Nicole L; Sutherland, Laura L; Scearce, Richard M; Balachandran, Harikrishnan; Bradley, Todd; Goodman, Derrick; Eaton, Amanda; Stanfield-Oakley, Sherry A; Tartaglia, James; Phogat, Sanjay; Pantaleo, Giuseppe; Esteban, Mariano; Gomez, Carmen E; Perdiguero, Beatriz; Jacobs, Bertram; Kibler, Karen; Korber, Bette; Montefiori, David C; Ferrari, Guido; Vandergrift, Nathan; Liao, Hua-Xin; Tomaras, Georgia D; Haynes, Barton F

    2018-04-15

    A preventive human immunodeficiency virus type 1 (HIV-1) vaccine is an essential part of the strategy to eradicate AIDS. A critical question is whether antibodies that do not neutralize primary isolate (tier 2) HIV-1 strains can protect from infection. In this study, we investigated the ability of an attenuated poxvirus vector (NYVAC) prime-envelope gp120 boost to elicit potentially protective antibody responses in a rhesus macaque model of mucosal simian-human immunodeficiency virus (SHIV) infection. NYVAC vector delivery of a group M consensus envelope, trivalent mosaic envelopes, or a natural clade B isolate B.1059 envelope elicited antibodies that mediated neutralization of tier 1 viruses, cellular cytotoxicity, and phagocytosis. None of the macaques made neutralizing antibodies against the tier 2 SHIV SF162P3 used for mucosal challenge. Significant protection from infection was not observed for the three groups of vaccinated macaques compared to unvaccinated macaques, although binding antibody to HIV-1 Env correlated with decreased viremia after challenge. Thus, NYVAC Env prime-gp120 boost vaccination elicited polyfunctional, nonneutralizing antibody responses with minimal protective activity against tier 2 SHIV mucosal challenge. IMPORTANCE The antibody responses that confer protection against HIV-1 infection remain unknown. Polyfunctional antibody responses correlated with time to infection in previous macaque studies. Determining the ability of vaccines to induce these types of responses is critical for understanding how to improve upon the one efficacious human HIV-1 vaccine trial completed thus far. We characterized the antibody responses induced by a NYVAC-protein vaccine and determined the protective capacity of polyfunctional antibody responses in an R5, tier 2 mucosal SHIV infection model. Copyright © 2018 American Society for Microbiology.

  17. Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

    PubMed

    Kooijman, Lotte J; Mapes, Samantha M; Pusterla, Nicola

    2016-07-01

    Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations. © 2016 The Author(s).

  18. Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

    PubMed Central

    Silva, Maria C.; Yu, Qian-Chun; Enquist, Lynn; Shenk, Thomas

    2003-01-01

    The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm. PMID:12970444

  19. A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

    PubMed Central

    Agnihothram, Sudhakar; Yount, Boyd L.; Donaldson, Eric F.; Huynh, Jeremy; Menachery, Vineet D.; Gralinski, Lisa E.; Graham, Rachel L.; Becker, Michelle M.; Tomar, Sakshi; Scobey, Trevor D.; Osswald, Heather L.; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K.; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R.; Baric, Ralph S.

    2014-01-01

    ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. PMID:24667706

  20. Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions.

    PubMed

    Read, G Sullivan; Patterson, Mary

    2007-02-01

    The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

  1. Recovery from severe novel coronavirus infection.

    PubMed

    Albarrak, Ali M; Stephens, Gwen M; Hewson, Roger; Memish, Ziad A

    2012-12-01

    We describe the third confirmed case of novel coronavirus infection in a resident of the Arabian Peninsula. Our patient presented, as did 2 prior cases, with severe pneumonia and renal dysfunction requiring intensive care support including assisted ventilation. However, unlike the earlier cases, and despite underlying chronic disease and a single kidney, he survived his infection and has been discharged home. The Ministry of Health continues active surveillance for additional cases. As this case report goes to press, 2 additional confirmed cases have been identified in Riyadh, Saudi Arabia. Contact investigations are in progress. Future work will focus not only on the origin of the virus and mechanisms of transmission, but also the host factors that influence pathogenesis and prognosis.

  2. Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013.

    PubMed

    Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Reusken, Chantal B E M; Ritz, Daniel; Godeke, Gert-Jan; Lattwein, Erik; Kallies, Stephan; Siemens, Artem; van Beek, Janko; Drexler, Jan F; Muth, Doreen; Bosch, Berend-Jan; Wernery, Ulrich; Koopmans, Marion P G; Wernery, Renate; Drosten, Christian

    2014-04-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.

  3. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  4. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    PubMed

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  5. The search for a structural basis for therapeutic intervention against the SARS coronavirus

    NASA Astrophysics Data System (ADS)

    Bartlam, M.; Xue, X.; Rao, Z.

    2008-01-01

    The severe acute respiratory syndrome (SARS) coronavirus outbreak in 2003 had profound social and economic impacts worldwide. This review highlights the importance of structural biology and shows that structures for drug design can be rapidly determined in the event of an emerging infectious disease.

  6. The structure of common-envelope remnants

    NASA Astrophysics Data System (ADS)

    Hall, Philip D.

    2015-05-01

    We investigate the structure and evolution of the remnants of common-envelope evolution in binary star systems. In a common-envelope phase, two stars become engulfed in a gaseous envelope and, under the influence of drag forces, spiral to smaller separations. They may merge to form a single star or the envelope may be ejected to leave the stars in a shorter period orbit. This process explains the short orbital periods of many observed binary systems, such as cataclysmic variables and low-mass X-ray binary systems. Despite the importance of these systems, and of common-envelope evolution to their formation, it remains poorly understood. Specifically, we are unable to confidently predict the outcome of a common-envelope phase from the properties at its onset. After presenting a review of work on stellar evolution, binary systems, common-envelope evolution and the computer programs used, we describe the results of three computational projects on common-envelope evolution. Our work specifically relates to the methods and prescriptions which are used for predicting the outcome. We use the Cambridge stellar-evolution code STARS to produce detailed models of the structure and evolution of remnants of common-envelope evolution. We compare different assumptions about the uncertain end-of-common envelope structure and envelope mass of remnants which successfully eject their common envelopes. In the first project, we use detailed remnant models to investigate whether planetary nebulae are predicted after common-envelope phases initiated by low-mass red giants. We focus on the requirement that a remnant evolves rapidly enough to photoionize the nebula and compare the predictions for different ideas about the structure at the end of a common-envelope phase. We find that planetary nebulae are possible for some prescriptions for the end-of-common envelope structure. In our second contribution, we compute a large set of single-star models and fit new formulae to the core radii of

  7. Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

    PubMed

    Berto, A; Anh, P H; Carrique-Mas, J J; Simmonds, P; Van Cuong, N; Tue, N T; Van Dung, N; Woolhouse, M E; Smith, I; Marsh, G A; Bryant, J E; Thwaites, G E; Baker, S; Rabaa, M A

    2018-02-01

    Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

  8. Envelopes in eclipsing binary stars

    NASA Technical Reports Server (NTRS)

    Huang, S.

    1972-01-01

    Theoretical research on eclipsing binaries is presented. The specific areas of investigation are the following: (1) the relevance of envelopes to the study of the light curves of eclipsing binaries, (2) the disk envelope, and (3) the spherical envelope.

  9. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang, Xuhua; Hew, Choy Leong, E-mail: dbshewcl@nus.edu.sg

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals ofmore » SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.« less

  10. LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

    PubMed Central

    Gu, Mingyu; LaJoie, Dollie; Chen, Opal S.; von Appen, Alexander; Ladinsky, Mark S.; Redd, Michael J.; Nikolova, Linda; Bjorkman, Pamela J.; Sundquist, Wesley I.; Ullman, Katharine S.; Frost, Adam

    2017-01-01

    Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope. PMID:28242692

  11. The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity.

    PubMed

    Raivio, Tracy L; Leblanc, Shannon K D; Price, Nancy L

    2013-06-01

    The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance.

  12. Phosphatidylinositol 4-Kinase IIIβ Is Required for Severe Acute Respiratory Syndrome Coronavirus Spike-mediated Cell Entry*

    PubMed Central

    Yang, Ning; Ma, Ping; Lang, Jianshe; Zhang, Yanli; Deng, Jiejie; Ju, Xiangwu; Zhang, Gongyi; Jiang, Chengyu

    2012-01-01

    Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection. PMID:22253445

  13. Middle East respiratory syndrome coronavirus: current situation and travel-associated concerns.

    PubMed

    Al-Tawfiq, Jaffar A; Omrani, Ali S; Memish, Ziad A

    2016-06-01

    The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. More than 1500 MERS-CoV cases were recorded in 42 months with a case fatality rate (CFR) of 40%. Meanwhile, 8000 cases of SARS-CoV were confirmed in six months with a CFR of 10%. The clinical presentation of MERS-CoV ranges from mild and non-specific presentation to progressive and severe pneumonia. No predictive signs or symptoms exist to differentiate MERS-CoV from community-acquired pneumonia in hospitalized patients. An apparent heterogeneity was observed in transmission. Most MERS-CoV cases were secondary to large outbreaks in healthcare settings. These cases were secondary to community-acquired cases, which may also cause family outbreaks. Travel-associated MERS infection remains low. However, the virus exhibited a clear tendency to cause large outbreaks outside the Arabian Peninsula as exemplified by the outbreak in the Republic of Korea. In this review, we summarize the current knowledge about MERS-CoV and highlight travel-related issues.

  14. Pancreatitis and Systemic Coronavirus Infection in a Ferret (Mustela putorius furo).

    PubMed

    Wills, Sarah E; Beaufrère, Hugues H; Brisson, Brigitte A; Fraser, Russell S; Smith, Dale A

    2018-06-01

    A 1-y-old spayed female ferret (Mustela putorius furo) was referred for additional diagnostic evaluation after physical examination by the referring veterinarian revealed a cranial abdominal mass. The ferret had a 2-wk history of inappetence, weight loss, and lethargy. On presentation, the ferret was thin, and an approximately 3-cm mass was palpable in the cranial abdomen. No other abnormalities were noted. Abdominal ultrasonography confirmed the presence of a soft-tissue structure, with a moderate blood supply and mesenteric lymphadenopathy. Fine-needle aspirates of the mass were nondiagnostic. Exploratory laparotomy revealed multiple nodules and thickened tissues throughout the mesentery, a thickened and nodular pancreas, and a small amount of free abdominal fluid. Histopathology of mesenteric, lymphatic, and pancreatic biopsies revealed suppurative pancreatitis and necrotizing and pyogranulomatous mesenteric steatitis. Positive immunohistochemistry for feline coronavirus confirmed a diagnosis of ferret systemic coronavirus disease (FSCD). The ferret was treated medically with oral prednisolone, improved dramatically, and was still doing well 22 mo after diagnosis. Although FSCD has been reported extensively, this case is noteworthy for the presence of suppurative pancreatitis and the positive long-term outcome after corticosteroid therapy.

  15. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

    PubMed

    Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

    2012-12-11

    A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is

  16. CD-loop Extension in Zika Virus Envelope Protein Key for Stability and Pathogenesis.

    PubMed

    Gallichotte, Emily N; Dinnon, Kenneth H; Lim, Xin-Ni; Ng, Thiam-Seng; Lim, Elisa X Y; Menachery, Vineet D; Lok, Shee-Mei; Baric, Ralph S

    2017-12-05

    With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  17. Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody.

    PubMed

    Sultana, Hameeda; Foellmer, Harald G; Neelakanta, Girish; Oliphant, Theodore; Engle, Michael; Ledizet, Michel; Krishnan, Manoj N; Bonafé, Nathalie; Anthony, Karen G; Marasco, Wayne A; Kaplan, Paul; Montgomery, Ruth R; Diamond, Michael S; Koski, Raymond A; Fikrig, Erol

    2009-07-01

    West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

  18. Purification and characterization of zebrafish hatching enzyme - an evolutionary aspect of the mechanism of egg envelope digestion.

    PubMed

    Sano, Kaori; Inohaya, Keiji; Kawaguchi, Mari; Yoshizaki, Norio; Iuchi, Ichiro; Yasumasu, Shigeki

    2008-12-01

    There are two hatching enzyme homologues in the zebrafish genome: zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT-PCR analysis revealed that ZHE1 was mainly expressed in pre-hatching embryos, whereas ZHE2 was rarely expressed. This was consistent with the results obtained in an experiment conducted at the protein level, which demonstrated that one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid. Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes, medaka high choriolytic enzyme (MHCE) and medaka low choriolytic enzyme (MLCE), which cooperatively digest the egg envelope. The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. The cross-species digestion using enzymes and substrates of zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the same sites of the egg envelope proteins of two species, suggesting that the substrate specificity of ZHE1 is quite similar to that of MHCE. However, MLCE did not show such similarity. Because HCE and LCE are the result of gene duplication in the evolutionary pathway of Teleostei, the present study suggests that ZHE1 and MHCE maintain the character of an ancestral hatching enzyme, and that MLCE acquires a new function, such as promoting the complete digestion of the egg envelope swollen by MHCE.

  19. CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

    PubMed Central

    Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

    2000-01-01

    The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

  20. CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

    PubMed

    Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

    2000-07-01

    The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

  1. Clinical, hematological, and biochemical findings in puppies with coronavirus and parvovirus enteritis

    PubMed Central

    Castro, Tatiana X.; Cubel Garcia, Rita de Cássia N.; Gonçalves, Luciana P. S.; Costa, Erika M.; Marcello, Gracy C.G.; Labarthe, Norma V.; Mendes-de-Almeida, Flavya

    2013-01-01

    The clinical and laboratory findings in puppies naturally infected with canine coronavirus (CCoV) and/or canine parvovirus (CPV) were compared with findings in uninfected puppies. Lymphopenia was the only parameter related to CCoV infection that was statistically significant; vomiting, anorexia, lethargy, hemorrhagic fluid diarrhea, leukopenia, lymphopenia, thrombocytopenia, hypoglycemia, and hypoproteinemia were correlated with CPV infection. PMID:24155496

  2. Acute Middle East Respiratory Syndrome Coronavirus Infection in Livestock Dromedaries, Dubai, 2014

    PubMed Central

    Corman, Victor M.; Wong, Emily Y.M.; Tsang, Alan K.L.; Muth, Doreen; Lau, Susanna K. P.; Khazanehdari, Kamal; Zirkel, Florian; Ali, Mansoor; Nagy, Peter; Juhasz, Jutka; Wernery, Renate; Joseph, Sunitha; Syriac, Ginu; Elizabeth, Shyna K.; Patteril, Nissy Annie Georgy; Woo, Patrick C. Y.; Drosten, Christian

    2015-01-01

    Camels carry Middle East respiratory syndrome coronavirus, but little is known about infection age or prevalence. We studied >800 dromedaries of all ages and 15 mother–calf pairs. This syndrome constitutes an acute, epidemic, and time-limited infection in camels <4 years of age, particularly calves. Delayed social separation of calves might reduce human infection risk. PMID:25989145

  3. A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    López-Igual, Rocío; Lechno-Yossef, Sigal; Fan, Qing; Herrero, Antonia; Wolk, C. Peter

    2012-01-01

    Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O2) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox− phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox− mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the PglnA promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep. PMID:22753066

  4. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

    PubMed

    Rogers, Jason V; Rose, Mark D

    2014-12-02

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

  5. Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

    PubMed Central

    Rogers, Jason V.; Rose, Mark D.

    2014-01-01

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. PMID:25467943

  6. The autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be functionally substituted by rachiplusia ou multiple nucleopolyhedrovirus ODV-E56

    USDA-ARS?s Scientific Manuscript database

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of viral ho...

  7. NKp44 receptor mediates interaction of the envelope glycoproteins from the West-Nile and dengue viruses with Natural Killer cells

    PubMed Central

    Hershkovitz, Oren; Rosental, Benyamin; Rosenberg, Lior Ann; Navarro-Sanchez, Martha Erika; Jivov, Sergey; Zilka, Alon; Gershoni-Yahalom, Orly; Brient-Litzler, Elodie; Bedouelle, Hugues; Ho, Joanna W.; Campbell, Kerry S.; Rager-Zisman, Bracha; Despres, Philippe; Porgador, Angel

    2009-01-01

    Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve natural killer (NK) cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally-mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein (EIII); it also binds to WNV virus-like particles (VLPs). These WNV-VLPs and WNV-EIII directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK de-granulation. Finally, WNV infection of cells results in increased binding of recombinant NKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFNγ secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 antibodies. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein. PMID:19635919

  8. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flavivirusesmore » and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.« less

  9. Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human

    PubMed Central

    Song, Huai-Dong; Tu, Chang-Chun; Zhang, Guo-Wei; Wang, Sheng-Yue; Zheng, Kui; Lei, Lian-Cheng; Chen, Qiu-Xia; Gao, Yu-Wei; Zhou, Hui-Qiong; Xiang, Hua; Zheng, Hua-Jun; Chern, Shur-Wern Wang; Cheng, Feng; Pan, Chun-Ming; Xuan, Hua; Chen, Sai-Juan; Luo, Hui-Ming; Zhou, Duan-Hua; Liu, Yu-Fei; He, Jian-Feng; Qin, Peng-Zhe; Li, Ling-Hui; Ren, Yu-Qi; Liang, Wen-Jia; Yu, Ye-Dong; Anderson, Larry; Wang, Ming; Xu, Rui-Heng; Wu, Xin-Wei; Zheng, Huan-Ying; Chen, Jin-Ding; Liang, Guodong; Gao, Yang; Liao, Ming; Fang, Ling; Jiang, Li-Yun; Li, Hui; Chen, Fang; Di, Biao; He, Li-Juan; Lin, Jin-Yan; Tong, Suxiang; Kong, Xiangang; Du, Lin; Hao, Pei; Tang, Hua; Bernini, Andrea; Yu, Xiao-Jing; Spiga, Ottavia; Guo, Zong-Ming; Pan, Hai-Yan; He, Wei-Zhong; Manuguerra, Jean-Claude; Fontanet, Arnaud; Danchin, Antoine; Niccolai, Neri; Li, Yi-Xue; Wu, Chung-I; Zhao, Guo-Ping

    2005-01-01

    The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002–2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003. PMID:15695582

  10. Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane.

    PubMed

    Taniyama, Toshiyuki; Tsuda, Natsumi; Sueda, Shinji

    2018-06-15

    The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

  11. Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

    NASA Astrophysics Data System (ADS)

    Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

    2006-08-01

    Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

  12. Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

    PubMed Central

    Vogel, Liesbeth; Van der Lubben, Mariken; Te Lintelo, Eddie G.; Bekker, Cornelis P.J.; Geerts, Tamara; Schuijff, Leontine S.; Grinwis, Guy C.M.; Egberink, Herman F.; Rottier, Peter J.M.

    2010-01-01

    Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed. PMID:20663472

  13. Role of the nuclear envelope in the pathogenesis of age-related bone loss and osteoporosis

    PubMed Central

    Vidal, Christopher; Bermeo, Sandra; Fatkin, Diane; Duque, Gustavo

    2012-01-01

    The nuclear envelope is the most important border in the eukaryotic cell. The role of the nuclear envelope in cell differentiation and function is determined by a constant interaction between the elements of the nuclear envelope and the transcriptional regulators involved in signal transcription pathways. Among those components of the nuclear envelope, there is a growing evidence that changes in the expression of A-type lamins, which are essential components of the nuclear lamina, are associated with age-related changes in bone affecting the capacity of differentiation of mesenchymal stem cells into osteoblasts, favoring adipogenesis and affecting the function and survival of the osteocytes. Overall, as A-type lamins are considered as the 'guardians of the soma', these proteins are also essential for the integrity and quality of the bone and pivotal for the longevity of the musculoskeletal system. PMID:23951459

  14. Development of chemical inhibitors of the SARS coronavirus: viral helicase as a potential target.

    PubMed

    Keum, Young-Sam; Jeong, Yong-Joo

    2012-11-15

    Severe acute respiratory syndrome (SARS) was the first pandemic in the 21st century to claim more than 700 lives worldwide. However, effective anti-SARS vaccines or medications are currently unavailable despite being desperately needed to adequately prepare for a possible SARS outbreak. SARS is caused by a novel coronavirus, and one of its components, a viral helicase, is emerging as a promising target for the development of chemical SARS inhibitors. In the following review, we describe the characterization, family classification, and kinetic movement mechanisms of the SARS coronavirus (SCV) helicase-nsP13. We also discuss the recent progress in the identification of novel chemical inhibitors of nsP13 in the context of our recent discovery of the strong inhibition of the SARS helicase by natural flavonoids, myricetin and scutellarein. These compounds will serve as important resources for the future development of anti-SARS medications. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. [An electron microscopy study of the structure of polyelectrolyte microcapsules containing protein and containing no protein].

    PubMed

    Kazakova, L I; Dubrovskiĭ, A V; Moshkov, D A; Shabarchina, L I; Sukhorukov, B I

    2007-01-01

    Electron micrographs of ultrathin sections of polyelectrolyte microparticles containing protein and free from protein for the formation of which CaCO3 spherulites served as a core basis have been obtained and analyzed. Polyelectrolyte microparticles with the number of alternately layered polyelectrolyte layers of polystyrene sulfonate and polyallylamine from 6 to 11 have been studied. It follows from the data obtained that protein-free polyelectrolyte particles having the dimensions 4.5-5 mm are formations of an intricate internal organization, which consist of a set of threadlike and closed nanoelements of polyelectrolyte nature with a thickness of 20-30 nm. The particles containing six to eight polyelectrolyte layers lack the external envelope; therefore, they were called polyelectrolyte microspherulites. With the number of layers nine and more, when a polyelectrolyte envelope appears on the surface, they transfer into polyelectrolyte microcapsules. It was found that, in a protein-containing polyelectrolyte microcapsule, as distinct from protein-free polyelectrolyte microspherulite and microcapsule, polyelectrolytes are located only in the nearsurface layer, and the external spatially organized envelope restricts the internal volume filled with protein solution. As the number of polyelectrolyte layers increases, the thickness of the envelope increases. The reasons for such substantial differences in the structures of polyelectrolyte microcapsules formed on protein-containing and protein-free CaCO3 spherulite are discussed.

  16. Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling.

    PubMed

    Zhang, Qingzhan; Yoo, Dongwan

    2016-12-02

    Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are emerged and reemerging viruses in pigs, and together with transmissible gastroenteritis virus (TGEV), pose significant economic concerns to the swine industry. These viruses infect epithelial cells of the small intestine and cause watery diarrhea, dehydration, and a high mortality in neonatal piglets. Type I interferons (IFN-α/β) are major antiviral cytokines forming host innate immunity, and in turn, these enteric coronaviruses have evolved to modulate the host innate immune signaling during infection. Accumulating evidence however suggests that IFN induction and signaling in the intestinal epithelial cells differ from other epithelial cells, largely due to distinct features of the gut epithelial mucosal surface and commensal microflora, and it appears that type III interferon (IFN-λ) plays a key role to maintain the antiviral state in the gut. This review describes the recent understanding on the immune evasion strategies of porcine enteric coronaviruses and the role of different types of IFNs for intestinal antiviral innate immunity. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Antigenic properties of the envelope of influenza virus rendered soluble by surfactant-solvent systems

    PubMed Central

    Larin, N. M.; Gallimore, P. H.

    1971-01-01

    Dissociating chemical treatments employing surfactant-solvent systems were applied to purified influenza A and B viruses to obtain viral preparations possessing a significantly higher or lower haemagglutinating activity than the intact virus. All preparations, whether with high or low haemagglutinating activity, with the exception of envelope protein solubilized by Triton X-100, were significantly lacking in the ability to excite the formation of haemagglutination-inhibiting and virus-neutralizing antibodies in inoculated ferrets. In contrast to other treatments, Triton X-100 treatment of virus significantly enhanced the antigenicity of viral protein as judged by virus neutralization and haemagglutination inhibition tests. Yet the haemagglutinating activity of the envelope protein solubilized with Triton X-100 was about 1% that of the intact virus. Results suggest that the correlation assumed to exist between the haemagglutinating activity of influenza virus and its ability to excite the formation of humoral antibodies is coincidental. Another important point is that the specific antigenicity of viral protein may be lost or enhanced owing to effects, other than solubilization, by surface-active agents. PMID:5291750

  18. Cortical processing of dynamic sound envelope transitions.

    PubMed

    Zhou, Yi; Wang, Xiaoqin

    2010-12-08

    Slow envelope fluctuations in the range of 2-20 Hz provide important segmental cues for processing communication sounds. For a successful segmentation, a neural processor must capture envelope features associated with the rise and fall of signal energy, a process that is often challenged by the interference of background noise. This study investigated the neural representations of slowly varying envelopes in quiet and in background noise in the primary auditory cortex (A1) of awake marmoset monkeys. We characterized envelope features based on the local average and rate of change of sound level in envelope waveforms and identified envelope features to which neurons were selective by reverse correlation. Our results showed that envelope feature selectivity of A1 neurons was correlated with the degree of nonmonotonicity in their static rate-level functions. Nonmonotonic neurons exhibited greater feature selectivity than monotonic neurons in quiet and in background noise. The diverse envelope feature selectivity decreased spike-timing correlation among A1 neurons in response to the same envelope waveforms. As a result, the variability, but not the average, of the ensemble responses of A1 neurons represented more faithfully the dynamic transitions in low-frequency sound envelopes both in quiet and in background noise.

  19. Origin, differentiation and functional ultrastructure of egg envelopes in the cestode Echinococcus multilocularis Leuckart, 1863 (Cyclophyllidea: Taeniidae).

    PubMed

    Świderski, Zdzisław; Miquel, Jordi; Azzouz-Maache, Samira; Pétavy, Anne-Françoise

    2017-07-01

    The origin, differentiation and functional ultrastructure of oncospheral or egg envelopes in Echinococcus multilocularis Leuckart, 1863 were studied by transmission electron microscopy (TEM) and cytochemistry. The purpose of our study is to describe the formation of the four primary embryonic envelopes, namely vitelline capsule, outer envelope, inner envelope and oncospheral membrane, and their transformation into the oncospheral or egg envelopes surrounding the mature hexacanth. This transformation takes place in the preoncospheral phase of embryonic development. The vitelline capsule and oncospheral membrane are thin membranes, while the outer and inner envelopes are thick cytoplasmic layers formed by two specific types of blastomeres: the outer envelope by cytoplasmic fusion of two macromeres and the inner envelope by cytoplasmic fusion of three mesomeres. Both outer and inner envelopes are therefore cellular in origin and syncytial in nature. During the advanced phase of embryonic development, the outer and inner envelopes undergo great modifications. The outer envelope remains as a metabolically active layer involved in the storage of glycogen and lipids for the final stages of egg development and survival. The inner envelope is the most important protective layer because of its thick layer of embryophoric blocks that assures oncospheral protection and survival. This embryophore is the principal layer of mature eggs, affording physical and physiological protection for the differentiated embryo or oncosphere, since the outer envelope is stripped from the egg before it is liberated. The embryophore is very thick and impermeable, consisting of polygonal blocks of an inert keratin-like protein held together by a cementing substance. The embryophore therefore assures extreme resistance of eggs, enabling them to withstand a wide range of environmental temperatures and physicochemical conditions.

  20. Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

    PubMed Central

    van de Ven, W J; Vermorken, A J; Onnekink, C; Bloemers, H P; Bloemendal, H

    1978-01-01

    A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction. Images PMID:702639

  1. Human endogenous retrovirus-FRD envelope protein (syncytin 2) expression in normal and trisomy 21-affected placenta.

    PubMed

    Malassiné, André; Frendo, Jean-Louis; Blaise, Sandra; Handschuh, Karen; Gerbaud, Pascale; Tsatsaris, Vassilis; Heidmann, Thierry; Evain-Brion, Danièle

    2008-01-23

    Human trophoblast expresses two fusogenic retroviral envelope proteins, the widely studied syncytin 1, encoded by HERV-W and the recently characterized syncytin 2 encoded by HERV-FRD. Here we studied syncytin 2 in normal and Trisomy 21-affected placenta associated with abnormal trophoblast differentiation. Syncytin 2 immunolocalization was restricted throughout normal pregnancy to some villous cytotrophoblastic cells (CT). During the second trimester of pregnancy, syncytin 2 was immunolocalized in some cuboidal CT in T21 placentas, whereas in normal placentas it was observed in flat CT, extending into their cytoplasmic processes. In vitro, CT isolated from normal placenta fuse and differentiate into syncytiotrophoblast. At the same time, syncytin 2 transcript levels decreased significantly with syncytiotrophoblast formation. In contrast, CT isolated from T21-affected placentas fused and differentiated poorly and no variation in syncytin 2 transcript levels was observed. Syncytin 2 expression illustrates the abnormal trophoblast differentiation observed in placenta of fetal T21-affected pregnancies.

  2. Human endogenous retrovirus-FRD envelope protein (syncytin 2) expression in normal and trisomy 21-affected placenta

    PubMed Central

    Malassiné, André; Frendo, Jean-Louis; Blaise, Sandra; Handschuh, Karen; Gerbaud, Pascale; Tsatsaris, Vassilis; Heidmann, Thierry; Evain-Brion, Danièle

    2008-01-01

    Human trophoblast expresses two fusogenic retroviral envelope proteins, the widely studied syncytin 1, encoded by HERV-W and the recently characterized syncytin 2 encoded by HERV-FRD. Here we studied syncytin 2 in normal and Trisomy 21-affected placenta associated with abnormal trophoblast differentiation. Syncytin 2 immunolocalization was restricted throughout normal pregnancy to some villous cytotrophoblastic cells (CT). During the second trimester of pregnancy, syncytin 2 was immunolocalized in some cuboidal CT in T21 placentas, whereas in normal placentas it was observed in flat CT, extending into their cytoplasmic processes. In vitro, CT isolated from normal placenta fuse and differentiate into syncytiotrophoblast. At the same time, syncytin 2 transcript levels decreased significantly with syncytiotrophoblast formation. In contrast, CT isolated from T21-affected placentas fused and differentiated poorly and no variation in syncytin 2 transcript levels was observed. Syncytin 2 expression illustrates the abnormal trophoblast differentiation observed in placenta of fetal T21-affected pregnancies. PMID:18215254

  3. Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response▿

    PubMed Central

    Glowacka, Ilona; Bertram, Stephanie; Müller, Marcel A.; Allen, Paul; Soilleux, Elizabeth; Pfefferle, Susanne; Steffen, Imke; Tsegaye, Theodros Solomon; He, Yuxian; Gnirss, Kerstin; Niemeyer, Daniela; Schneider, Heike; Drosten, Christian; Pöhlmann, Stefan

    2011-01-01

    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion. PMID:21325420

  4. Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop.

    PubMed

    Chabierski, Stefan; Barzon, Luisa; Papa, Anna; Niedrig, Matthias; Bramson, Jonathan L; Richner, Justin M; Palù, Giorgio; Diamond, Michael S; Ulbert, Sebastian

    2014-05-09

    West Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II. We tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies. Using this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity. Recombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies

  5. A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    PubMed Central

    Balboni, Andrea; Gallina, Laura; Palladini, Alessandra; Prosperi, Santino; Battilani, Mara

    2012-01-01

    Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population. PMID:22654650

  6. Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines

    PubMed Central

    Miura, Tanya A.; Wang, Jieru; Holmes, Kathryn V.; Mason, Robert J.

    2007-01-01

    We analyzed the ability of two rat coronavirus (RCoV) strains, sialodacryoadenitis virus (SDAV) and Parker’s RCoV (RCoV-P), to infect rat alveolar type I cells and induce chemokine expression. Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCoV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemoattractants. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation. PMID:17804032

  7. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctionalmore » region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.« less

  8. Nonstationary envelope process and first excursion probability

    NASA Technical Reports Server (NTRS)

    Yang, J.

    1972-01-01

    A definition of the envelope of nonstationary random processes is proposed. The establishment of the envelope definition makes it possible to simulate the nonstationary random envelope directly. Envelope statistics, such as the density function, joint density function, moment function, and level crossing rate, which are relevent to analyses of catastrophic failure, fatigue, and crack propagation in structures, are derived. Applications of the envelope statistics to the prediction of structural reliability under random loadings are discussed in detail.

  9. Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

    PubMed

    Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

    2017-03-01

    Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Detection of Severe Acute Respiratory Syndrome-Like, Middle East Respiratory Syndrome-Like Bat Coronaviruses and Group H Rotavirus in Faeces of Korean Bats.

    PubMed

    Kim, H K; Yoon, S-W; Kim, D-J; Koo, B-S; Noh, J Y; Kim, J H; Choi, Y G; Na, W; Chang, K-T; Song, D; Jeong, D G

    2016-08-01

    Bat species around the world have recently been recognized as major reservoirs of several zoonotic viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah virus and Hendra virus. In this study, consensus primer-based reverse transcriptase polymerase chain reactions (RT-PCRs) and high-throughput sequencing were performed to investigate viruses in bat faecal samples collected at 11 natural bat habitat sites from July to December 2015 in Korea. Diverse coronaviruses were first detected in Korean bat faeces, including alphacoronaviruses, SARS-CoV-like and MERS-CoV-like betacoronaviruses. In addition, we identified a novel bat rotavirus belonging to group H rotavirus which has only been described in human and pigs until now. Therefore, our results suggest the need for continuing surveillance and additional virological studies in domestic bat. © 2016 Blackwell Verlag GmbH.

  11. The roles of transportation and transportation hubs in the propagation of influenza and coronaviruses: a systematic review.

    PubMed

    Browne, Annie; Ahmad, Sacha St-Onge; Beck, Charles R; Nguyen-Van-Tam, Jonathan S

    2016-01-01

    Respiratory viruses spread in humans across wide geographical areas in short periods of time, resulting in high levels of morbidity and mortality. We undertook a systematic review to assess the evidence that air, ground and sea mass transportation systems or hubs are associated with propagating influenza and coronaviruses. Healthcare databases and sources of grey literature were searched using pre-defined criteria between April and June 2014. Two reviewers screened all identified records against the protocol, undertook risk of bias assessments and extracted data using a piloted form. Results were analysed using a narrative synthesis. Forty-one studies met the eligibility criteria. Risk of bias was high in the observational studies, moderate to high in the reviews and moderate to low in the modelling studies. In-flight influenza transmission was identified substantively on five flights with up to four confirmed and six suspected secondary cases per affected flight. Five studies highlighted the role of air travel in accelerating influenza spread to new areas. Influenza outbreaks aboard cruise ships affect 2-7% of passengers. Influenza transmission events have been observed aboard ground transport vehicles. High heterogeneity between studies and the inability to exclude other sources of infection means that the risk of influenza transmission from an index case to other passengers cannot be accurately quantified. A paucity of evidence was identified describing severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus transmission events associated with transportation systems or hubs. Air transportation appears important in accelerating and amplifying influenza propagation. Transmission occurs aboard aeroplanes, at the destination and possibly at airports. Control measures to prevent influenza transmission on cruise ships are needed to reduce morbidity and mortality. There is no recent evidence of sea transport accelerating influenza

  12. Inactivation and safety testing of Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Kumar, Mia; Mazur, Steven; Ork, Britini L.; Postnikova, Elena; Hensley, Lisa E.; Jahrling, Peter B.; Johnson, Reed; Holbrook, Michael R.

    2015-01-01

    Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a recently emerged virus that has caused a number of human infections and deaths, primarily in the Middle East. The transmission of MERS-CoV to humans has been proposed to be as a result of contact with camels, but evidence of human-to-human transmission also exists. In order to work with MERS-CoV in a laboratory setting, the US Centers for Disease Control and Prevention (CDC) has determined that MERS-CoV should be handled at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Many processes and procedures used to characterize MERS-CoV and to evaluate samples from MERS-CoV infected animals are more easily and efficiently completed at BSL-2 or lower containment. In order to complete experimental work at BSL-2, demonstration or proof of inactivation is required before removal of specimens from biocontainment laboratories. In the studies presented here, we evaluated typical means of inactivating viruses prior to handling specimens at a lower biocontainment level. We found that Trizol, AVL buffer and gamma irradiation were effective at inactivating MERS-CoV, that formaldehyde-based solutions required at least 30 minutes of contact time in a cell culture system while a mixture of methanol and acetone required 60 minutes to inactivate MERS-CoV. Together, these data provide a foundation for safely inactivating MERS-CoV, and potentially other coronaviruses, prior to removal from biocontainment facilities. PMID:26190637

  13. Requirement of cholesterol in the viral envelope for dengue virus infection.

    PubMed

    Carro, Ana C; Damonte, Elsa B

    2013-06-01

    The role of cholesterol in the virus envelope or in the cellular membranes for dengue virus (DENV) infection was examined by depletion with methyl-beta-cyclodextrin (MCD) or nystatin. Pretreatment of virions with MCD or nystatin significantly reduced virus infectivity in a dose-dependent manner. By contrast, pre-treatment of diverse human cell lines with MCD or nystatin did not affect DENV infection. The four DENV serotypes were similarly inactivated by cholesterol-extracting drugs and infectivity was partially rescued when virion suspensions were treated with MCD in the presence of bovine serum. The addition of serum or exogenous water-soluble cholesterol after MCD treatment did not produce a reversion of MCD inactivating effect. Furthermore, virion treatment with extra cholesterol exerted also a virucidal effect. Binding and uptake of cholesterol-deficient DENV into the host cell were not impaired, whereas the next step of fusion between virion envelope and endosome membrane leading to virion uncoating and release of nucleocapsids to the cytoplasm appeared to be prevented, as determined by the retention of capsid protein in cells infected with MCD inactivated-DENV virions. Thereafter, the infection was almost completely inhibited, given the failure of viral RNA synthesis and viral protein expression in cells infected with MCD-treated virions. These data suggest that envelope cholesterol is a critical factor in the fusion process for DENV entry. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. The persistent prevalence and evolution of cross-family recombinant coronavirus GCCDC1 among a bat population: a two-year follow-up.

    PubMed

    Obameso, Joseph O; Li, Hong; Jia, Hao; Han, Min; Zhu, Shiyan; Huang, Canping; Zhao, Yuhui; Zhao, Min; Bai, Yu; Yuan, Fei; Zhao, Honglan; Peng, Xia; Xu, Wen; Tan, Wenjie; Zhao, Yingze; Yuen, Kwok-Yung; Liu, William J; Lu, Lin; Gao, George F

    2017-12-01

    Bats are connected with the increasing numbers of emerging and re-emerging viruses that may break the species barrier and spread into the human population. Coronaviruses are one of the most common viruses discovered in bats, which were considered as the natural source of recent human-susceptible coronaviruses, i.e. SARS-COV and MERS-CoV. Our previous study reported the discovery of a bat-derived putative cross-family recombinant coronavirus with a reovirus gene p10, named as Ro-BatCoV GCCDC1. In this report, through a two-year follow-up of a special bat population in one specific cave of south China, we illustrate that Ro-BatCoV GCCDC1 persistently circulates among bats. Notably, through the longitudinal observation, we identified the dynamic evolution of Ro-BatCoV GCCDC1 in bats represented by continuously recombination events. Our study provides the first glimpse of the virus evolution in one longitudinally observed bat population cohort and underlines the surveillance and pre-warning of potential interspecies transmittable viruses in bats.

  15. Mutations in the Vaccinia Virus A33R and B5R Envelope Proteins That Enhance Release of Extracellular Virions and Eliminate Formation of Actin-Containing Microvilli without Preventing Tyrosine Phosphorylation of the A36R Protein

    PubMed Central

    Katz, Ehud; Ward, Brian M.; Weisberg, Andrea S.; Moss, Bernard

    2003-01-01

    The spread of vaccinia virus in cell cultures is mediated by virions that adhere to the tips of specialized actin-containing microvilli and also by virions that are released into the medium. The use of a small plaque-forming A36R gene deletion mutant to select spontaneous second-site mutants exhibiting enhanced virus release was described previously. Two types of mutations were found: C-terminal truncations of the A33R envelope protein and a single amino acid substitution of the B5R envelope protein. In the present study, we transferred each type of mutation into a wild-type virus background in order to study their effects in vitro and in vivo. The two new mutants conserved the enhanced virus release properties of the original isolates; the A33R mutant produced considerably more extracellular virus than the B5R mutant. The extracellular virus particles contained the truncated A33R protein in one case and the mutated B5R protein in the other. Remarkably, both mutants failed to form actin tails and specialized microvilli, despite the presence of an intact A36R gene. The synthesis of the A36R protein as well as its physical association with the mutated or wild-type A33R protein was demonstrated. Moreover, the A36R protein was tyrosine phosphorylated, a step mediated by a membrane-associated Src kinase that regulates the nucleation of actin polymerization. The presence of large numbers of adherent virions on the cell surface argued against rapid dissociation as having a key role in preventing actin tail formation. Thus, the A33R and B5R proteins may be more directly involved in the formation or stabilization of actin tails than had been previously thought. When mice were inoculated intranasally, the A33R mutant was highly attenuated and the B5R mutant was mildly attenuated compared to wild-type virus. Enhanced virus release, therefore, did not compensate for the loss of actin tails and specialized microvilli. PMID:14581563

  16. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses.

    PubMed

    Su, Shuo; Wong, Gary; Shi, Weifeng; Liu, Jun; Lai, Alexander C K; Zhou, Jiyong; Liu, Wenjun; Bi, Yuhai; Gao, George F

    2016-06-01

    Human coronaviruses (HCoVs) were first described in the 1960s for patients with the common cold. Since then, more HCoVs have been discovered, including those that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), two pathogens that, upon infection, can cause fatal respiratory disease in humans. It was recently discovered that dromedary camels in Saudi Arabia harbor three different HCoV species, including a dominant MERS HCoV lineage that was responsible for the outbreaks in the Middle East and South Korea during 2015. In this review we aim to compare and contrast the different HCoVs with regard to epidemiology and pathogenesis, in addition to the virus evolution and recombination events which have, on occasion, resulted in outbreaks amongst humans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

    PubMed

    Dubayle, Jean; Vialle, Sandrine; Schneider, Diane; Pontvianne, Jérémy; Mantel, Nathalie; Adam, Olivier; Guy, Bruno; Talaga, Philippe

    2015-03-10

    Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    PubMed Central

    Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

    2016-01-01

    Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

  19. LINCing complex functions at the nuclear envelope

    PubMed Central

    Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

    2013-01-01

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

  20. Seasonal benefits of a natural propolis envelope to honey bee immunity and colony health.

    PubMed

    Borba, Renata S; Klyczek, Karen K; Mogen, Kim L; Spivak, Marla

    2015-11-01

    Honey bees, as social insects, rely on collective behavioral defenses that produce a colony-level immune phenotype, or social immunity, which in turn impacts the immune response of individuals. One behavioral defense is the collection and deposition of antimicrobial plant resins, or propolis, in the nest. We tested the effect of a naturally constructed propolis envelope within standard beekeeping equipment on the pathogen and parasite load of large field colonies, and on immune system activity, virus and storage protein levels of individual bees over the course of a year. The main effect of the propolis envelope was a decreased and more uniform baseline expression of immune genes in bees during summer and autumn months each year, compared with the immune activity in bees with no propolis envelope in the colony. The most important function of the propolis envelope may be to modulate costly immune system activity. As no differences were found in levels of bacteria, pathogens and parasites between the treatment groups, the propolis envelope may act directly on the immune system, reducing the bees' need to activate the physiologically costly production of humoral immune responses. Colonies with a natural propolis envelope had increased colony strength and vitellogenin levels after surviving the winter in one of the two years of the study, despite the fact that the biological activity of the propolis diminished over the winter. A natural propolis envelope acts as an important antimicrobial layer enshrouding the colony, benefiting individual immunity and ultimately colony health. © 2015. Published by The Company of Biologists Ltd.

  1. Recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection.

    PubMed

    Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

    2007-02-01

    We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.

  2. Recombinant Truncated Nucleocapsid Protein as Antigen in a Novel Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Severe Acute Respiratory Syndrome Coronavirus Infection▿

    PubMed Central

    Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

    2007-01-01

    We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection. PMID:17202310

  3. Novel coronaviruses, astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China.

    PubMed

    Han, H-J; Wen, H-L; Zhao, L; Liu, J-W; Luo, L-M; Zhou, C-M; Qin, X-R; Zhu, Y-L; Liu, M-M; Qi, R; Li, W-Q; Yu, H; Yu, X-J

    2017-12-01

    Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long-footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat-borne viruses. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

  4. Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

    PubMed Central

    Rycaj, Kiera; Plummer, Joshua B.; Li, Ming; Yin, Bingnan; Frerich, Katherine; Garza, Jeremy G.; Shen, Jianjun; Lin, Kevin; Yan, Peisha; Glynn, Sharon A.; Dorsey, Tiffany H.; Hunt, Kelly K.; Ambs, Stefan; Johanning, Gary L.

    2012-01-01

    Background The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. Methods We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb–mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. Results The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti–HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm3; difference = 972.89 mm3, 95% CI = 470.17 to 1475.61 mm3; P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb–treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of

  5. Airway Memory CD4(+) T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses.

    PubMed

    Zhao, Jincun; Zhao, Jingxian; Mangalam, Ashutosh K; Channappanavar, Rudragouda; Fett, Craig; Meyerholz, David K; Agnihothram, Sudhakar; Baric, Ralph S; David, Chella S; Perlman, Stanley

    2016-06-21

    Two zoonotic coronaviruses (CoVs)-SARS-CoV and MERS-CoV-have crossed species to cause severe human respiratory disease. Here, we showed that induction of airway memory CD4(+) T cells specific for a conserved epitope shared by SARS-CoV and MERS-CoV is a potential strategy for developing pan-coronavirus vaccines. Airway memory CD4(+) T cells differed phenotypically and functionally from lung-derived cells and were crucial for protection against both CoVs in mice. Protection was dependent on interferon-γ and required early induction of robust innate and virus-specific CD8(+) T cell responses. The conserved epitope was also recognized in SARS-CoV- and MERS-CoV-infected human leukocyte antigen DR2 and DR3 transgenic mice, indicating potential relevance in human populations. Additionally, this epitope was cross-protective between human and bat CoVs, the progenitors for many human CoVs. Vaccine strategies that induce airway memory CD4(+) T cells targeting conserved epitopes might have broad applicability in the context of new CoVs and other respiratory virus outbreaks. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhardwaj, Kanchan; Palaninathan, Satheesh; Alcantara, Joanna Maria Ortiz

    2008-03-31

    The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involvedmore » in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.« less

  7. Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique

    Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependentmore » phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion.

  8. STS-8 postal Stamp envelope

    NASA Technical Reports Server (NTRS)

    1983-01-01

    STS-8 postal Stamp envelope with Challenger insignia, USA eagle stamp, 25th NASA anniversary stamp. The envelope is stamped with various postmarks, one saying Kennedy Space Center, Fl., another saying 'Returned to earth, Edwards AFB, CA'.

  9. Role of the Phosphatidylserine Receptor TIM-1 in Enveloped-Virus Entry

    PubMed Central

    Moller-Tank, Sven; Kondratowicz, Andrew S.; Davey, Robert A.; Rennert, Paul D.

    2013-01-01

    The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence. PMID:23698310

  10. Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers†

    PubMed Central

    Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Churchill, Michael J.; Kim, Alice M.; Kuhn, Peter; Dawson, Philip; Moulton, Hong M.; Bestwick, Richard K.; Iversen, Patrick L.; Buchmeier, Michael J.

    2005-01-01

    The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5′ untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection. PMID:16014928

  11. Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

    PubMed

    Abuhammad, Areej; Al-Aqtash, Rua'a A; Anson, Brandon J; Mesecar, Andrew D; Taha, Mutasem O

    2017-11-01

    The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CL pro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CL pro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CL pro . HKU4-CoV 3CL pro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CL pro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CL pro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CL pro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CL pro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CL pro . MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CL pro is a potential drug target for coronavirus infection. HKU4-CoV 3CL pro is a useful surrogate model for the identification of MERS-CoV 3CL pro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CL pro inhibitors

  12. Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

    PubMed

    Liu, Guan-Ting; Kung, Hsiu-Ni; Chen, Chung-Kuan; Huang, Cheng; Wang, Yung-Li; Yu, Cheng-Pu; Lee, Chung-Pei

    2018-02-26

    Although a vesicular nucleocytoplasmic transport system is believed to exist in eukaryotic cells, the features of this pathway are mostly unknown. Here, we report that the BFRF1 protein of the Epstein-Barr virus improves vesicular transport of nuclear envelope (NE) to facilitate the translocation and clearance of nuclear components. BFRF1 expression induces vesicles that selectively transport nuclear components to the cytoplasm. With the use of aggregation-prone proteins as tools, we found that aggregated nuclear proteins are dispersed when these BFRF1-induced vesicles are formed. BFRF1-containing vesicles engulf the NE-associated aggregates, exit through from the NE, and putatively fuse with autophagic vacuoles. Chemical treatment and genetic ablation of autophagy-related factors indicate that autophagosome formation and autophagy-linked FYVE protein-mediated autophagic proteolysis are involved in this selective clearance of nuclear proteins. Remarkably, vesicular transport, elicited by BFRF1, also attenuated nuclear aggregates accumulated in neuroblastoma cells. Accordingly, induction of NE-derived vesicles by BFRF1 facilitates nuclear protein translocation and clearance, suggesting that autophagy-coupled transport of nucleus-derived vesicles can be elicited for nuclear component catabolism in mammalian cells.-Liu, G.-T., Kung, H.-N., Chen, C.-K., Huang, C., Wang, Y.-L., Yu, C.-P., Lee, C.-P. Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

  13. Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo.

    PubMed

    Moulton, H M; Fletcher, S; Neuman, B W; McClorey, G; Stein, D A; Abes, S; Wilton, S D; Buchmeier, M J; Lebleu, B; Iversen, P L

    2007-08-01

    The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

  14. HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

    Cancer.gov

    The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that Env is the only viral protein expressed on HIV’s surface, knowing its detailed structure—especially in the unbound state—may be critical for designing antibodies and vaccines against HIV.

  15. NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

    PubMed Central

    Simmons, T.; Heywood, P.; Hodge, L.

    1973-01-01

    The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G1) has been investigated in synchronized HeLa S3 cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G1 was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope. PMID:4752403

  16. Marine derived compounds as binders of the White spot syndrome virus VP28 envelope protein: In silico insights from molecular dynamics and binding free energy calculations.

    PubMed

    Sivakumar, K C; Sajeevan, T P; Bright Singh, I S

    2016-10-01

    White spot syndrome virus (WSSV) remains as one of the most dreadful pathogen of the shrimp aquaculture industry owing to its high virulence. The cumulative mortality reaches up to 100% within in 2-10days in a shrimp farm. Currently, no chemotherapeutics are available to control WSSV. The viral envelope protein, VP28, located on the surface of the virus particle acts as a vital virulence factor in the initial phases of inherent WSSV infection in shrimp. Hence, inhibition of envelope protein VP28 could be a novel way to deal with infection by inhibiting its interaction in the endocytic pathway. In this direction, a timely attempt was made to recognize a potential drug candidate of marine origin against WSSV using VP28 as a target by employing in silico docking and molecular dynamic simulations. A virtual library of 388 marine bioactive compounds was extracted from reports published in Marine Drugs. The top ranking compounds from docking studies were chosen from the flexible docking based on the binding affinities (ΔGb). In addition, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions. The results suggested that the two compounds obtained a negative binding free energy with -40.453kJ/mol and -31.031kJ/mol for compounds with IDs 30797199 and 144162 respectively. The RMSD curve indicated that 30797199 moves into the hydrophobic core, while the position of 144162 atoms changes abruptly during simulation and is mostly stabilized by water bridges. The shift in RMSD values of VP28 corresponding to ligand RMSD gives an insight into the ligand induced conformational changes in the protein. This study is first of its kind to elucidate the explicit binding of chemical inhibitor to WSSV major structural protein VP28. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

    PubMed

    Gardner, Thomas J; Stein, Kathryn R; Duty, J Andrew; Schwarz, Toni M; Noriega, Vanessa M; Kraus, Thomas; Moran, Thomas M; Tortorella, Domenico

    2016-12-14

    The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

  18. HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

    Cancer.gov

    The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that

  19. Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

    PubMed

    Guimaraes, Ana M S; Brandão, Paulo E; de Moraes, Wanderlei; Cubas, Zalmir S; Santos, Leonilda C; Villarreal, Laura Y B; Robes, Rogério R; Coelho, Fabiana M; Resende, Mauricio; Santos, Renata C F; Oliveira, Rosangela C; Yamaguti, Mauricio; Marques, Lucas M; Neto, Renata L; Buzinhani, Melissa; Marques, Regina; Messick, Joanne B; Biondo, Alexander W; Timenetsky, Jorge

    2009-06-01

    A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

  20. Creating a Lunar EVA Work Envelope

    NASA Technical Reports Server (NTRS)

    Griffin, Brand N.; Howard, Robert; Rajulu, Sudhakar; Smitherman, David

    2009-01-01

    A work envelope has been defined for weightless Extravehicular Activity (EVA) based on the Space Shuttle Extravehicular Mobility Unit (EMU), but there is no equivalent for planetary operations. The weightless work envelope is essential for planning all EVA tasks because it determines the location of removable parts, making sure they are within reach and visibility of the suited crew member. In addition, using the envelope positions the structural hard points for foot restraints that allow placing both hands on the job and provides a load path for reacting forces. EVA operations are always constrained by time. Tasks are carefully planned to ensure the crew has enough breathing oxygen, cooling water, and battery power. Planning first involves computers using a virtual work envelope to model tasks, next suited crew members in a simulated environment refine the tasks. For weightless operations, this process is well developed, but planetary EVA is different and no work envelope has been defined. The primary difference between weightless and planetary work envelopes is gravity. It influences anthropometry, horizontal and vertical mobility, and reaction load paths and introduces effort into doing "overhead" work. Additionally, the use of spacesuits other than the EMU, and their impacts on range of motion, must be taken into account. This paper presents the analysis leading to a concept for a planetary EVA work envelope with emphasis on lunar operations. There is some urgency in creating this concept because NASA has begun building and testing development hardware for the lunar surface, including rovers, habitats and cargo off-loading equipment. Just as with microgravity operations, a lunar EVA work envelope is needed to guide designers in the formative stages of the program with the objective of avoiding difficult and costly rework.