Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

PubMed Central

Teoh, Pak-Guan; Huang, Zhi-Shun; Pong, Wen-Li; Chen, Po-Chiang

2014-01-01

ABSTRACT The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal α2 and α4 helices of each monomer. Pairing of α2-α2′ creates a continuous hydrophobic surface, while the α4-α4′ helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the α4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the α4-α4′ helix pair conformation is critical in preserving the overall α-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the α4-α4′ helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. IMPORTANCE Structure-based mutagenesis study of the dengue virus core protein (CP) reveals that the α4-α4′ helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus

Sequence of events in measles virus replication: role of phosphoprotein-nucleocapsid interactions.

PubMed

Brunel, Joanna; Chopy, Damien; Dosnon, Marion; Bloyet, Louis-Marie; Devaux, Patricia; Urzua, Erica; Cattaneo, Roberto; Longhi, Sonia; Gerlier, Denis

2014-09-01

The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor. Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten

Analysis of Hantaan Virus RNA: Evidence for a New Genus of Bunyaviridae

DTIC Science & Technology

1983-01-01

accomplished by et aL, 1976). This characteristic distin- incubation of 50 pg of virion RNA with 50 guishes bunyaviruses from rhabdoviruses #Ci of cytidine 3...to that of mM HEPES, 8 mM dithiothreitol, 10 mM nucleocapsids from the rhabdovirus , ve- MI 3s, and 5 sM ATP (Sigma). Following sicular stomatitis...similar to those described for the bunya- +DNas 11,606 virus Uukuniemi (Ranki and Pettersson, +1 paM CT 5864 1975) or for the rhabdovirus VSV (Huang +5

Structure and assembly of the Ebola virus nucleocapsid

PubMed Central

Wan, William; Kolesnikova, Larissa; Clarke, Mairi; Koehler, Alexander; Noda, Takeshi; Becker, Stephan; Briggs, John A. G.

2017-01-01

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause hemorrhagic fever1. Filoviruses are within the order Mononegavirales2 which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein (NP) and other viral proteins to form a helical nucleocapsid (NC). NC acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into NP-NP interactions have been derived from structural studies of oligomerized, RNA-encapsidating NP3–6 and cryo-electron microscopy (cryo-EM) of NC7–12 or NC-like structures11–13. There have been no high-resolution reconstructions of complete mononegavirus NCs. Here, we have applied cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus NC within intact viruses and recombinant NC-like assemblies. These structures reveal the identity and arrangement of the NC components, and suggest that the formation of an extended alpha-helix from the disordered C-terminal region of NP-core links NP oligomerization, NC condensation, RNA encapsidation, and accessory protein recruitment. PMID:29144446

[Prevalence of antibodies to hantavirus among hemodialysis patients with end-stage renal failure in Kaunas and its district].

PubMed

Dargevicius, Arvydas; Petraityte, Rasa; Sribikiene, Birute; Sileikiene, Elvyra; Razukeviciene, Loreta; Ziginskiene, Edita; Vorobjoviene, Rita; Razanskiene, Ausra; Sasnauskas, Kestutis; Bumblyte, Inga Arūne; Kuzminskis, Vytautas

2007-01-01

The objective of this study was to investigate the prevalence of antibodies to hantaviruses among hemodialysis patients with end-stage renal failure in Kaunas and its district. Serums of 218 patients from four dialysis centers of Kaunas district were tested by using the immunoglobulin G antibody-capture enzyme-linked immunosorbent assay (ELISA). The reactivity of ELISA-positive sera was proven in Western blot tests using various hantavirus recombinant nucleocapsid proteins. The yeast-expressed nucleocapsid proteins were used for testing. Antibodies against Dobrava/Hantaan and Puumala hantaviruses were found in 16 patients (seroprevalence 7.4%). Most of the sera were positive for Dobrava hantavirus (81%). The ratio of males to females was 1.2:1. Seroprevalence was significantly higher in older patients. Results indicate that antibodies to two hantaviruses (Dobrava/Hantaan virus and Puumala virus) are prevalent among hemodialysis patients in Kaunas district with approximately the same seroprevalence as in neighboring countries.

Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Slibinskas, Rimantas; Staniulis, Juozas; Sasnauskas, Kestutis; Shiell, Brian J; Wang, Lin-Fa; Michalski, Wojtek P

2007-03-01

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

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

Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-10-15

Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particlesmore » had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.« less

Quantification of RNA Content in Reconstituted Ebola Virus Nucleocapsids by Immunoprecipitation.

PubMed

Banadyga, Logan; Ebihara, Hideki

2017-01-01

Immunoprecipitations are commonly used to isolate proteins or protein complexes and assess protein-protein interactions; however, they can also be used to assess protein-RNA complexes. Here we describe an adapted RNA immunoprecipitation technique that permits the quantification of RNA content in Ebola virus nucleocapsids that have been reconstituted in vitro by transient transfection.

Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

PubMed

Dolnik, Olga; Kolesnikova, Larissa; Welsch, Sonja; Strecker, Thomas; Schudt, Gordian; Becker, Stephan

2014-10-01

Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the

Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction.

PubMed

Bhella, David; Ralph, Adam; Yeo, Robert Paul

2004-07-02

Measles virus is a highly contagious virus that, despite the existence of an effective vaccine, is a major cause of illness and mortality worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form a helical ribonucleoprotein complex known as the nucleocapsid. This structure serves as the template for both transcription and replication. Paramyxovirus nucleocapsids are flexible structures, a trait that has hitherto hampered structural analysis even at low resolution. We have investigated the extent of this structural plasticity, using real-space methods to calculate three-dimensional reconstructions of recombinant nucleocapsids from cryo-negative stain transmission electron micrographs. Images of short sections of helix were sorted according to both pitch (the axial rise per turn) and twist (the number of subunits per turn). Our analysis indicates that there is extensive conformational flexibility within these structures, ranging in pitch from 50 Angstrom to 66 Angstrom, while twist varies from at least 13.04 to 13.44 with a greater number of helices comprising around 13.1 subunits per turn. We have also investigated the influence of the C terminus of N on helix conformation, analysing nucleocapsids after having removed this domain by trypsin digestion. We have found that this causes a marked change in both pitch and twist, such that the pitch becomes shorter, ranging from 46 Angstrom to 52 Angstrom, while more helices have a twist of approximately 13.3 subunits per turn. Our findings lead us to propose a mechanism whereby changes in conformation, influenced by interactions between viral or host proteins and the C terminus of N, might have a role in regulating the balance of transcription and replication during virus infection.

Role of nucleocapsid protein of hantaviruses in intracellular traffic of viral glycoproteins.

PubMed

Shimizu, Kenta; Yoshimatsu, Kumiko; Koma, Takaaki; Yasuda, Shumpei P; Arikawa, Jiro

2013-12-26

To understand the role of nucleocapsid protein (NP) of hantaviruses in viral assembly, the effect of NP on intracellular traffic of viral glycoproteins Gn and Gc was investigated. Double staining of viral and host proteins in Hantaan virus (HTNV)-infected Vero E6 cells showed that Gn and Gc were localized to cis-Golgi, in which virus particles are thought to be formed. When HTNV Gn and Gc were expressed by a plasmid encoding glycoprotein precursor (GPC), which is posttranslationally cleaved into Gn and Gc, Gn was localized to cis-Golgi, whereas Gc showed diffuse distribution in the cytoplasm in 32.9% of Gc-positive cells. The ratio of the diffused Gc-positive cells was significantly decreased to 15.0% by co-expression of HTNV NP. Co-expression of HTNV GPC with NPs of other hantaviruses, such as Seoul virus, Puumala virus and Sin Nombre virus, also reduced the ratios of diffused Gc-positive cells to 13.5%, 25.2%, and 11.6%, respectively. Among amino- and carboxyl-terminally truncated HTNV NPs, NP75-429, NP116-429, NP1-333, NP1-233, and NP1-155 possessed activity to reduce the ratio of diffused Gc-positive cells, while NP155-429 and NP1-116 did not. NP30-429 has partial activity. These results indicate that amino acid region 116-155 of NP is important for the activity, although amino acid region 1-30 is partially related. Truncation of the HTNV Gc cytoplasmic tail caused an increase in diffused Gc-positive cells. In addition, the effect of coexpression of HTNV NP was weakened. These results suggest that HTNV NP has a role to promote Golgi localization of Gc through a mechanism possibly mediated by the Gc cytoplasmic tail. Copyright © 2013 Elsevier B.V. All rights reserved.

Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle

PubMed Central

Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan

2016-01-01

Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

PubMed

Bulavaitė, Aistė; Lasickienė, Rita; Tamošiūnas, Paulius Lukas; Simanavičius, Martynas; Sasnauskas, Kęstutis; Žvirblienė, Aurelija

2017-04-01

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

PubMed

Griot-Wenk, M E; Cherpillod, P; Koch, A; Zurbriggen, R; Bruckner, L; Wittek, R; Zurbriggen, A

2001-06-01

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

PubMed

Banadyga, Logan; Hoenen, Thomas; Ambroggio, Xavier; Dunham, Eric; Groseth, Allison; Ebihara, Hideki

2017-08-09

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

PubMed

Dzagurova, T K; Solopova, O N; Sveshnikov, P G; Korotina, N A; Balovneva, M V; Leonovich, O A; Varlamov, N E; Malkin, G A; Sotskova, S E; Tkachenko, E A

2013-01-01

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

Detection of links between Ebola nucleocapsid and virulence using disorder analysis.

PubMed

Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

2015-08-01

The underlying reasons for the differences in the virulence of various types of Ebola virus (EBOV) remain unknown. Comparison of the percentage of disorder (PID) in nucleocapsid proteins VP30 and NP reveals high correlation between nucleocapsid PIDs and the case-fatality rates of EBOV. The higher disorder of these proteins is likely to be needed for more efficient multiplication of virus copies via more efficient viral RNA transcription and more promiscuous protein binding potential. This is important for the more efficient assistance of nucleocapsid in viral particle budding and of the assembly and mobility of viral proteins across the host membrane and within the cytoplasm. A more comprehensive knowledge of the molecular mechanisms of EBOV virulence would also lead to new and more effective strategies in vaccine development.

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

PubMed Central

Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

2013-01-01

Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability.

PubMed

Jaru-Ampornpan, Peera; Jengarn, Juggragarn; Wanitchang, Asawin; Jongkaewwattana, Anan

2017-01-15

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDV AVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage

Genetic Diversity and Reassortment of Hantaan Virus Tripartite RNA Genomes in Nature, the Republic of Korea

PubMed Central

Kim, Jeong-Ah; Kim, Won-keun; No, Jin Sun; Lee, Seung-Ho; Lee, Sook-Young; Kim, Ji Hye; Kho, Jeong Hoon; Lee, Daesang; Song, Dong Hyun; Gu, Se Hun; Jeong, Seong Tae; Park, Man-Seong; Kim, Heung-Chul; Klein, Terry A.; Song, Jin-Won

2016-01-01

Background Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the most prevalent hantavirus in the Republic of Korea (ROK). It is the causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) in humans and maintained in the striped field mouse, Apodemus agrarius, the primary zoonotic host. Clinical HFRS cases have been reported commonly in HFRS-endemic areas of Gyeonggi province. Recently, the death of a member of the ROK military from Gangwon province due to HFRS prompted an investigation of the epidemiology and distribution of hantaviruses in Gangwon and Gyeonggi provinces that border the demilitarized zone separating North and South Korea. Methodology and Principal Findings To elucidate the geographic distribution and molecular diversity of HTNV, whole genome sequences of HTNV Large (L), Medium (M), and Small (S) segments were acquired from lung tissues of A. agrarius captured from 2003–2014. Consistent with the clinical incidence of HFRS established by the Korea Centers for Disease Control & Prevention (KCDC), the prevalence of HTNV in naturally infected mice in Gangwon province was lower than for Gyeonggi province. Whole genomic sequences of 34 HTNV strains were identified and a phylogenetic analysis showed geographic diversity of the virus in the limited areas. Reassortment analysis first suggested an occurrence of genetic exchange of HTNV genomes in nature, ROK. Conclusion/Significance This study is the first report to demonstrate the molecular prevalence of HTNV in Gangwon province. Whole genome sequencing of HTNV showed well-supported geographic lineages and the molecular diversity in the northern region of ROK due to a natural reassortment of HTNV genomes. These observations contribute to a better understanding of the genetic diversity and molecular evolution of hantaviruses. Also, the full-length of HTNV tripartite genomes will provide a database for phylogeographic analysis of spatial and temporal

Hantaan virus surveillance targeting small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005.

PubMed

Klein, Terry A; Kang, Hae Ji; Gu, Se Hun; Moon, Sungsil; Shim, So-Hee; Park, Yon Mi; Lee, Sook-Young; Kim, Heung-Chul; Chong, Sung-Tae; O'Guinn, Monica; Lee, John S; Turell, Michael J; Song, Jin-Won

2011-12-01

In response to a hemorrhagic fever with renal syndrome case in November 2000, a seasonal rodent-borne disease surveillance program was initiated at Dagmar North Training Area (DNTA), Gyeonggi Province, Republic of Korea. From April 2001-December 2005, 1,848 small mammals were captured. Apodemus agrarius accounted for 92.5%, followed by Mus musculus (3.6%), Crocidura lasiura (2.1%), and Microtus fortis (1.1%). Three species of rodents were found to be antibody-positive (Ab+) for Hantaan virus (HTNV): A. agrarius (22.3%), M. musculus (9.1%), and M. fortis (5.0%). Ab+ rates for A. agrarius increased with increasing weight (age), except for those weighing <10 g. The peak HTNV transmission period in Korea coincided with the peak reproductive potential of A. agrarius during the fall (August/September) surveys. HTNV strains from DNTA were distinct from HTNV strains from the People's Republic of China. From these studies, more accurate risk assessments can be developed to better protect personnel from rodent-borne diseases. © 2011 The Society for Vector Ecology.

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

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

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.

2008-08-15

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicatedmore » that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.« less

Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus.

PubMed

Jain, R K; Pandey, Amar N; Krishnareddy, M; Mandal, Bikash

2005-12-01

In vitro gene expression strategy was used for the production of polyclonal antiserum to the nucleocapsid protein (NP) of Groundnut bud necrosis virus (GBNV). The GBNV NP gene from cowpea isolate was cloned into 6x His-tagged UA cloning vector and expressed in Escherichia coli [M15] cells. The fusion protein was detected in insoluble fraction and was purified by using Ni-NTA agarose resin. The purified 6x His-fusion protein ( approximately 32 kDa) was used for immunisation to produce a high titre polyclonal antiserum. The antiserum to the NP of GBNV at 1:4000 dilution detected successfully natural infection of GBNV and Watermelon bud necrosis virus in a wide range of cucurbitaceous, leguminous and solanaceous hosts from different locations.

Muju virus, a novel hantavirus harboured by the arvicolid rodent Myodes regulus in Korea

PubMed Central

Song, Ki-Joon; Baek, Luck Ju; Moon, Sungsil; Ha, Si Jung; Kim, Sang Hyun; Park, Kwang Sook; Klein, Terry A.; Sames, William; Kim, Heung-Chul; Lee, John S.; Yanagihara, Richard; Song, Jin-Won

2008-01-01

Acute-phase sera from >5 % of cases of haemorrhagic fever with renal syndrome occurring annually in Korea have been found to exhibit a fourfold or higher antibody titre to Puumala virus (PUUV) than to Hantaan virus (HTNV) by double-sandwich IgM ELISA, suggesting the existence of a PUUV-related hantavirus. Based on the phylogenetic relationships among arvicolid rodents, the royal vole (Myodes regulus) was targeted as a likely reservoir host of hantavirus. Using RT-PCR, a genetically distinct hantavirus, designated Muju virus (MUJV), was detected in lung tissue of royal voles, captured in widely separated geographical regions in Korea during 1996–2007. Pairwise analysis of the full-length S (1857 nt) and M (3634 nt) segments of MUJV indicated approximately 77 % sequence similarity with PUUV. At the amino acid level, MUJV differed from PUUV by 5.5–6.9 % (nucleocapsid) and 10.0–11.6 % (Gn and Gc envelope glycoproteins). Interstrain variation of MUJV sequences from royal voles captured in different regions suggested geographic-specific clustering. Neutralizing antibody titres against PUUV were two- to sixfold higher than to HTNV in sera of MUJV-infected Myodes regulus. Although virus isolation attempts were unsuccessful, the collective data indicate that MUJV is a distinct hantavirus species. PMID:17947538

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

PubMed Central

Campagna, Matthew R.; Liu, Fei; Mao, Richeng; Mills, Courtney; Cai, Dawei; Guo, Fang; Zhao, Xuesen; Ye, Hong; Cuconati, Andrea; Guo, Haitao; Chang, Jinhong; Xu, Xiaodong; Block, Timothy M.

2013-01-01

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B. PMID:23576513

Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress.

PubMed

Biswas, Siddhartha; Willis, Leslie G; Fang, Minggang; Nie, Yingchao; Theilmann, David A

2018-02-01

During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV. IMPORTANCE Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin.

PubMed

Song, Tao; Fang, Liurong; Wang, Dang; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

2016-06-16

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Viral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and

Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4

PubMed Central

Jesus, Desyree Murta; Moussatche, Nissin; McFadden, Baron D.; Nielsen, Casey Paulasue; D’Costa, Susan M.; Condit, Richard C.

2015-01-01

Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid. PMID:25765002

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

PubMed

Cherpillod, P; Tipold, A; Griot-Wenk, M; Cardozo, C; Schmid, I; Fatzer, R; Schobesberger, M; Zurbriggen, R; Bruckner, L; Roch, F; Vandevelde, M; Wittek, R; Zurbriggen, A

2000-07-01

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

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

Lamb, Kristen; Lokesh, G.L.; Sherman, Michael

2010-10-25

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to themore » mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.« less

Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

PubMed Central

Cui, Xiuji; Ludgate, Laurie; Ning, Xiaojun

2013-01-01

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion. PMID:23966388

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

PubMed Central

Tellinghuisen, Timothy L.; Hamburger, Agnes E.; Fisher, Bonnie R.; Ostendorp, Ralf; Kuhn, Richard J.

1999-01-01

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly. PMID:10364277

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus.

PubMed

Lai, Qingying; Wu, Wenbi; Li, Ao; Wang, Wei; Yuan, Meijin; Yang, Kai

2018-05-01

Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K ( ac98 ) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene ( ac100 ) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus

PubMed Central

Lai, Qingying; Li, Ao; Wang, Wei; Yuan, Meijin

2018-01-01

ABSTRACT Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome

Development and Application of a Saccharomyces cerevisiae-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus

PubMed Central

Gibertoni, Aliandra M.; Montassier, Maria de Fátima S.; Sena, Janete A. D.; Givisiez, Patrícia E. N.; Furuyama, Cibele R. A. G.; Montassier, Hélio J.

2005-01-01

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. PMID:15815038

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

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

Raymond, Donald D.; Piper, Mary E.; Gerrard, Sonja R.

2010-07-13

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-{angstrom} crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA speciesmore » of {approx}100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous {approx}100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.« less

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

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

Liu Chao; Li Zhaofei; Wu Wenbi

2008-12-05

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc{sup ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc{sup ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are presentmore » in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.« less

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

PubMed

Milles, Sigrid; Jensen, Malene Ringkjøbing; Communie, Guillaume; Maurin, Damien; Schoehn, Guy; Ruigrok, Rob W H; Blackledge, Martin

2016-08-01

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats.

PubMed

Amada, Takako; Yoshimatsu, Kumiko; Yasuda, Shumpei P; Shimizu, Kenta; Koma, Takaaki; Hayashimoto, Nobuhito; Gamage, Chandika D; Nishio, Sanae; Takakura, Akira; Arikawa, Jiro

2013-10-01

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats. Copyright © 2013 Elsevier B.V. All rights reserved.

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

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

Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

2015-03-13

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two othermore » retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.« less

Serologic Cross-Reactions between Nucleocapsid Proteins of Human Respiratory Syncytial Virus and Human Metapneumovirus

PubMed Central

Zhang, Yange; Pohl, Jan; Brooks, W. Abdullah

2015-01-01

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) share virologic and epidemiologic features and cause clinically similar respiratory illness predominantly in young children. In a previous study of acute febrile respiratory illness in Bangladesh, we tested paired serum specimens from 852 children presenting fever and cough for diagnostic increases in titers of antibody to hRSV and hMPV by enzyme immunoassay (EIA). Unexpectedly, of 93 serum pairs that showed a ≥4-fold increase in titers of antibody to hRSV, 24 (25.8%) showed a concurrent increase in titers of antibody to hMPV; of 91 pairs showing an increase to hMPV, 13 (14.3%) showed a concurrent increase to hRSV. We speculated that common antigens shared by these viruses explain this finding. Since the nucleocapsid (N) proteins of these viruses show the greatest sequence homology, we tested hyperimmune antisera prepared for each virus against baculovirus-expressed recombinant N (recN) proteins for potential cross-reactivity. The antisera were reciprocally reactive with both proteins. To localize common antigenic regions, we first expressed the carboxy domain of the hMPV N protein that was the most highly conserved region within the hRSV N protein. Although reciprocally reactive with antisera by Western blotting, this truncated protein did not react with hMPV IgG-positive human sera by EIA. Using 5 synthetic peptides that spanned the amino-terminal portion of the hMPV N protein, we identified a single peptide that was cross-reactive with human sera positive for either virus. Antiserum prepared for this peptide was reactive with recN proteins of both viruses, indicating that a common immunoreactive site exists in this region. PMID:25740767

Serologic cross-reactions between nucleocapsid proteins of human respiratory syncytial virus and human metapneumovirus.

PubMed

Zhang, Yange; Pohl, Jan; Brooks, W Abdullah; Erdman, Dean D

2015-05-01

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) share virologic and epidemiologic features and cause clinically similar respiratory illness predominantly in young children. In a previous study of acute febrile respiratory illness in Bangladesh, we tested paired serum specimens from 852 children presenting fever and cough for diagnostic increases in titers of antibody to hRSV and hMPV by enzyme immunoassay (EIA). Unexpectedly, of 93 serum pairs that showed a ≥ 4-fold increase in titers of antibody to hRSV, 24 (25.8%) showed a concurrent increase in titers of antibody to hMPV; of 91 pairs showing an increase to hMPV, 13 (14.3%) showed a concurrent increase to hRSV. We speculated that common antigens shared by these viruses explain this finding. Since the nucleocapsid (N) proteins of these viruses show the greatest sequence homology, we tested hyperimmune antisera prepared for each virus against baculovirus-expressed recombinant N (recN) proteins for potential cross-reactivity. The antisera were reciprocally reactive with both proteins. To localize common antigenic regions, we first expressed the carboxy domain of the hMPV N protein that was the most highly conserved region within the hRSV N protein. Although reciprocally reactive with antisera by Western blotting, this truncated protein did not react with hMPV IgG-positive human sera by EIA. Using 5 synthetic peptides that spanned the amino-terminal portion of the hMPV N protein, we identified a single peptide that was cross-reactive with human sera positive for either virus. Antiserum prepared for this peptide was reactive with recN proteins of both viruses, indicating that a common immunoreactive site exists in this region. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

PubMed

Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

2015-10-01

Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development

Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

PubMed Central

Mühlberger, Elke; Lötfering, Beate; Klenk, Hans-Dieter; Becker, Stephan

1998-01-01

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication. PMID:9765419

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

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.

Hantaan virus surveillance targeting small mammals at nightmare range, a high elevation military training area, Gyeonggi Province, Republic of Korea.

PubMed

Klein, Terry A; Kim, Heung-Chul; Chong, Sung-Tae; Kim, Jeong-Ah; Lee, Sook-Young; Kim, Won-Keun; Nunn, Peter V; Song, Jin-Won

2015-01-01

Rodent-borne disease surveillance was conducted at Nightmare Range (NM-R), near the demilitarized zone in northeast Gyeonggi Province, Republic of Korea, to identify hemorrhagic fever with renal syndrome (HFRS) risks for a mountainous high-elevation (500 m) military training site. Monthly surveys were conducted from January 2008-December 2009. A total of 1,720 small mammals were captured belonging to the Orders Rodentia [Families, Sciuridae (1 species) and Muridae (7 species)] and Soricomorpha [Family, Soricidae (1species)]. Apodemus agrarius, the primary reservoir for Hantaan virus (HTNV), accounted for 89.9% (1,546) of all small mammals captured, followed by Myodes regulus (4.0%), Crocidura lasiura (3.9%), Micromys minutus (1.4%), Mus musculus (0.3%), Microtus fortis (0.2%), Apodemus peninsulae (0.2%), Tamias sibiricus (0.1%), and Rattus norvegicus (<0.1%). Three species were antibody-positive (Ab+) for hantaviruses: A. agrarius (8.2%), M. minutus (4.2%), and C. lasiura (1.5%). HTNV specific RNA was detected in 93/127 Ab+ A. agrarius, while Imjin virus specific RNA was detected in 1/1 Ab+ C. lasiura. Overall, hantavirus Ab+ rates for A. agrarius increased with weight (age) and were significantly higher among males (10.9%) than females (5.1%) (P<0.0001). High A. agrarius gravid rates during the fall (August-September) were associated with peak numbers of HFRS cases in Korea that followed high gravid rates. From 79 RT-PCR positive A. agrarius, 12 HTNV RNA samples were sequenced and compared phylogenetically based on a 320 nt sequence from the GC glycoprotein-encoding M segment. These results demonstrate that the HTNV isolates from NM-R are distinctly separated from HTNV isolated from the People's Republic of China. These studies provide for improved disease risk assessments that identify military activities, rodent HTNV rates, and other factors associated with the transmission of hantaviruses during field training exercises.

Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein

PubMed Central

Leonard, Vincent H. J.; Kohl, Alain; Osborne, Jane C.; McLees, Angela; Elliott, Richard M.

2005-01-01

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein. PMID:16189017

[Differences in oligomerization of nucleocapsid protein of epidemic human influenza A(H1N1), A(H1N2) and B viruses].

PubMed

Prokudina, E N; Semenova, N P; Chumakov, V M; Burtseva, E I; Slepushkin, A N

2003-01-01

A comparative analysis of involving the nucleocapsid protein (NP) into shaping-up of SDS-resistant oligomers was carried out presently in circulating epidemic strains of human influenza, viruses A and B. The study results of viral isolates obtained from clinical samples and recent standard strains revealed that the involvement of NP in the SDS-resistant oligomers, which are different in various subtypes of influenza A viruses. According to this sign, the human viruses A(9H3N2) are close to the avian ones, in which, as proved by us previously, virtually the entire NP transforms itself into the oligomers resistant to SDS. About 10-20% of NP are involved in shaping-up the virus influenza A(H1N1) of SDS-resistant oligomers. No SDS-resistant NP-oligomers were detected in influenza of type B. It is suggested that the prevalence of human viruses A(H3N2) in NP-oligomers are the peculiarities of NP structure and of the presence of the PB1 protein from avian influenza virus.

Development of polyclonal antibodies against nucleocapsid protein of watermelon silver mottle virus and their application to diagnostic.

PubMed

Wu, Z; Wang, W; Li, Y; Rao, X

2014-01-01

Watermelon silver mottle virus (WSMoV) is an emerging disease of cucurbit crops in South China. Production of high-quality antibodies is necessary for the development of serological methods for detection of this virus. The nucleocapsid protein (NP) gene of WSMoV was amplified from WSMoV-infected watermelon leaves by RT-PCR and cloned into vector pET-28a (+) for prokaryotic expression. After identification via enzyme digestion and sequencing, the recombinant clone was transformed into Escherichia coli Rosetta (DE3) for protein expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the WSMoV NP fusion protein was 34.1 kDa. The fusion protein was purified and used as antigen for the preparation of polyclonal antisera in rabbits. Results of indirect ELISA and western blot analysis showed that the antisera reacted specifically with WSMoV NP. In addition, sensitivity and specificity of the antisera were examined on a number of infected field samples by indirect ELISA. These findings will facilitate further immunological and serological studies of WSMoV. .

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable

BM61 of Bombyx mori nucleopolyhedrovirus: its involvement in the egress of nucleocapsids from the nucleus.

PubMed

Shen, Hongxing; Chen, Keping

2012-04-05

All lepidopteran baculovirus genomes sequenced encode a homolog of the Bombyx mori nucleopolyhedrovirus orf61 gene (Bm61). To determine the role of Bm61 in the baculoviral life cycle, we constructed a Bm61 knockout virus and characterized it in cells. We observed that the Bm61 deletion bacmid led to a defect in production of infectious budded virus (BV). Quantitative PCR analysis of BV in the media culturing the transfected cell indicated that BV was not produced due to Bm61 deletion. Electron microscope analysis showed that in the knockout of Bm61, nucleocapsids were not transported from the nucleus to the cytoplasm. From these results we concluded that BM61 is required in the BV pathway for the egress of nucleocapsids from the nucleus to the cytoplasm. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

PubMed

Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

2017-06-21

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus

NASA Technical Reports Server (NTRS)

Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.

[Production of the monoclonal antibodies to the rabies virus nucleoprotein].

PubMed

Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

2013-01-01

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

Autographa californica multiple nucleopolyhedrovirus PK-1 is essential for nucleocapsid assembly

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

Liang, Changyong, E-mail: cyliang@yzu.edu.cn; Li, Min; Dai, Xuejuan

2013-09-01

PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential inmore » regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly. - Highlights: • A pk-1 knockout AcMNPV failed to produce infectious progeny. • The pk-1 deletion did not affect viral DNA replication. • The catalytic domain of protein kinases (PKc) of PK-1 was essential to viral infectivity. • The kinase activity of PK-1 is essential in regulating viral propagation. • PK-1 appears to phosphorylate some viral proteins that are essential for DNA packaging to regulate nucleocapsid assembly.« less

The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation.

PubMed Central

Wahlberg, J M; Boere, W A; Garoff, H

1989-01-01

The budding and the fusion processes of the enveloped animal virus Semliki Forest virus serve the purpose of transporting its nucleocapsid, containing its genome, from the cytoplasm of an infected cell into that of an uninfected one. We show here that, in the infected cell, the viral membrane (spike) proteins p62 and E1 are organized as heterodimers which are very resistant to dissociation in acidic conditions. In contrast, the mature form of the heterodimer, E2E1, which is found in the virus particle and which is generated by proteolytic processing of p62, is very prone to dissociate upon treatment with mildly acidic buffers. We discuss the possibility that this difference in behavior of the intracellular precursor form and the mature form of the spike protein complex represents an important regulatory mechanism for the processes involving membrane binding around the nucleocapsid during budding and membrane release from the nucleocapsid at the stage of virus fusion. Images PMID:2479769

Mutations in the Basic Region of the Mason-Pfizer Monkey Virus Nucleocapsid Protein Affect Reverse Transcription, Genomic RNA Packaging, and the Virus Assembly Site.

PubMed

Dostálková, Alžběta; Kaufman, Filip; Křížová, Ivana; Kultová, Anna; Strohalmová, Karolína; Hadravová, Romana; Ruml, Tomáš; Rumlová, Michaela

2018-05-15

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K 16 NK 18 EK 20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription. IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse

Phylogenetic Relationship of Necoclí Virus to Other South American Hantaviruses (Bunyaviridae: Hantavirus).

PubMed

Montoya-Ruiz, Carolina; Cajimat, Maria N B; Milazzo, Mary Louise; Diaz, Francisco J; Rodas, Juan David; Valbuena, Gustavo; Fulhorst, Charles F

2015-07-01

The results of a previous study suggested that Cherrie's cane rat (Zygodontomys cherriei) is the principal host of Necoclí virus (family Bunyaviridae, genus Hantavirus) in Colombia. Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences in this study confirmed that Necoclí virus is phylogenetically closely related to Maporal virus, which is principally associated with the delicate pygmy rice rat (Oligoryzomys delicatus) in western Venezuela. In pairwise comparisons, nonidentities between the complete amino acid sequence of the nucleocapsid protein of Necoclí virus and the complete amino acid sequences of the nucleocapsid proteins of other hantaviruses were ≥8.7%. Likewise, nonidentities between the complete amino acid sequence of the glycoprotein precursor of Necoclí virus and the complete amino acid sequences of the glycoprotein precursors of other hantaviruses were ≥11.7%. Collectively, the unique association of Necoclí virus with Z. cherriei in Colombia, results of the Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences, and results of the pairwise comparisons of amino acid sequences strongly support the notion that Necoclí virus represents a novel species in the genus Hantavirus. Further work is needed to determine whether Calabazo virus (a hantavirus associated with Z. brevicauda cherriei in Panama) and Necoclí virus are conspecific.

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies.

PubMed

Xu, Gelin; Weber, Patrick; Hu, Qiaoling; Xue, Honggang; Audry, Laurent; Li, Chengping; Wu, Jie; Bourhy, Herve

2007-10-01

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

PubMed

Doležal, Michal; Hadravová, Romana; Kožíšek, Milan; Bednárová, Lucie; Langerová, Hana; Ruml, Tomáš; Rumlová, Michaela

2016-09-23

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

A retrospective analysis of sera collected by the Hemorrhagic Fever Commission during the Korean Conflict.

PubMed

LeDuc, J W; Ksiazek, T G; Rossi, C A; Dalrymple, J M

1990-11-01

More than 600 sera from 245 patients with a clinical diagnosis of hemorrhagic fever were preserved by the Hemorrhagic Fever Commission during the Korean Conflict, 1951-1954. These sera were tested for IgM- and IgG-specific antibodies to Hantaan virus by enzyme immunoassay and for hantaviral antigen by immunoassay; one serum from each patient was tested by plaque reduction neutralization using both Hantaan and Seoul viruses. Only 15 patients failed to develop antihantaviral antibodies; most sera contained high titered IgM antibody on admission, and all were IgM-seropositive by day 7 after onset. Attempts to detect hantaviral antigen were unsuccessful. All seropositive patients had highest plaque reduction neutralization titers to Hantaan virus, suggesting that this virus was responsible for the disease seen. These results confirm that hemorrhagic fever of the Korean Conflict was due to Hantaan virus and demonstrate that measurement of specific IgM antibody is the method of choice for diagnosis of acute disease.

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

PubMed

Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

2016-03-01

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs.

PubMed

Kulkarni, Diwakar D; Venkatesh, Govindarajalu; Tosh, Chakradhar; Patel, Priyanka; Mashoria, Anita; Gupta, Vandana; Gupta, Sourabh; D, Senthilkumar

2016-01-01

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella†

PubMed Central

Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

1980-01-01

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated. Images PMID:16789191

The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity.

PubMed

Ottmann, M; Gabus, C; Darlix, J L

1995-03-01

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity.

The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity.

PubMed Central

Ottmann, M; Gabus, C; Darlix, J L

1995-01-01

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity. PMID:7853517

Pathways of nucleopolyhedrosis virus infection in the gypsy moth, Lymantria dispar

Treesearch

K. S. Shields

1985-01-01

Gypsy moth nucleopolyhedrosis virus polyhedral inclusion bodies dissolve slowly in host digestive fluids, in vitro. Infectious viral material is in the hemocoel two hours after ingestion of inclusion bodies. Hemocytes produce and release nucleocapsids throughout the course of infection, but in the fat body, nearly all nucleocapsids are enveloped and...

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

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

Alayyoubi, Maher; Leser, George P.; Kors, Christopher A.

Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ~2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. In this paper, we have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closelymore » represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. Finally, this rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.« less

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

DOE PAGES

Alayyoubi, Maher; Leser, George P.; Kors, Christopher A.; ...

2015-03-23

Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ~2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. In this paper, we have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closelymore » represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. Finally, this rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.« less

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein-RNA complex.

PubMed

Alayyoubi, Maher; Leser, George P; Kors, Christopher A; Lamb, Robert A

2015-04-07

Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.

The RING domain and the L79 residue of Z protein are involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious chimeric arenavirus-like particles.

PubMed

Casabona, Juan Cruz; Levingston Macleod, Jesica M; Loureiro, Maria Eugenia; Gomez, Guillermo A; Lopez, Nora

2009-07-01

Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

PubMed

Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

2018-06-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which Ac

Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

PubMed Central

De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

1992-01-01

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents. Images PMID:1631144

Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

PubMed

De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

1992-07-15

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

PubMed

Shtanko, Olena; Watanabe, Shinji; Jasenosky, Luke D; Watanabe, Tokiko; Kawaoka, Yoshihiro

2011-04-01

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

PubMed

Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

The interferon response circuit in antiviral host defense.

PubMed

Haller, O; Weber, F

2009-01-01

Viruses have learned to multiply in the face of a powerful innate and adaptive immune response of the host. They have evolved multiple strategies to evade the interferon (IFN) system which would otherwise limit virus growth at an early stage of infection. IFNs induce the synthesis of a range of antiviral proteins which serve as cell-autonomous intrinsic restriction factors. For example, the dynamin-like MxA GTPase inhibits the multiplication of influenza and bunyaviruses (such as La Crosse virus, Hantaan virus, Rift Valley Fever virus, and Crimean-Congo hemorrhagic fever virus) by binding and sequestering the nucleocapsid protein into large perinuclear complexes. To overcome such intracellular restrictions, virulent viruses either inhibit IFN synthesis, bind and inactivate secreted IFN molecules, block IFN-activated signaling, or disturb the action of IFN-induced antiviral proteins. Many viruses produce specialized proteins to disarm the danger signal or express virulence genes that target members of the IFN regulatory factor family (IRFs) or components of the JAK-STAT signaling pathway. An alternative evasion strategy is based on extreme viral replication speed which out-competes the IFN response. The identification of viral proteins with IFN antagonistic functions has great implications for disease prevention and therapy. Virus mutants lacking IFN antagonistic properties represent safe yet highly immunogenic candidate vaccines. Furthermore, novel drugs intercepting viral IFN-antagonists could be used to disarm the viral intruders.

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

PubMed

Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus

NASA Technical Reports Server (NTRS)

Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.

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.

First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form.

PubMed

de Rocquigny, H; Ficheux, D; Gabus, C; Fournié-Zaluski, M C; Darlix, J L; Roques, B P

1991-10-31

The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

PubMed

Cui, Xiuji; Luckenbaugh, Laurie; Bruss, Volker; Hu, Jianming

2015-10-01

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can

Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle.

PubMed

Surtees, Rebecca; Dowall, Stuart D; Shaw, Amelia; Armstrong, Stuart; Hewson, Roger; Carroll, Miles W; Mankouri, Jamel; Edwards, Thomas A; Hiscox, Julian A; Barr, John N

2016-10-15

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease. Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which

Investigations of Hemorrhagic Fever with Renal Syndrome (HFRS) in Yugoslavia.

DTIC Science & Technology

1991-11-07

Hantaan virus, occurs in Korea, the People’s Republic of China and far eastern U.S.S.R., as well as in eastern Europe, including the Balcan countries...Seoul and Prospect Hill viruses (13,14). Very recently, the investigators et al. reported a close etiologic relation between HFRS in Korea, China ...HFRS patients in Korea and China , possessed high antibody titers to Hantaan and Seoul viruses with much lower reactivity to Puumala and Prospect Hill

Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

PubMed Central

Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

1999-01-01

The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

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

Generation and characterization of monoclonal antibodies against Rift Valley fever virus nucleoprotein.

PubMed

Fafetine, J M; Domingos, A; Antunes, S; Esteves, A; Paweska, J T; Coetzer, J A W; Rutten, V P M G; Neves, L

2013-11-01

Due to the unpredictable and explosive nature of Rift Valley fever (RVF) outbreaks, rapid and accurate diagnostic assays for low-resource settings are urgently needed. To improve existing diagnostic assays, monoclonal antibodies (MAbs) specific for the nucleocapsid protein of RVF virus (RVFV) were produced and characterized. Four IgG2a MAbs showed specific binding to denatured nucleocapsid protein, both from a recombinant source and from inactivated RVFV, in Western blot analysis and in an enzyme-linked immunosorbent assay (ELISA). Cross-reactivity with genetically related and non-related arboviruses including Bunyamwera and Calovo viruses (Bunyaviridae family), West Nile and Dengue-2 viruses (Flaviviridae family), and Sindbis and Chikungunya viruses (Togaviridae family) was not detected. These MAbs represent a useful tool for the development of rapid diagnostic assays for early recognition of RVF. © 2013 Blackwell Verlag GmbH.

Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

PubMed

Dolnik, Olga; Stevermann, Lea; Kolesnikova, Larissa; Becker, Stephan

2015-01-01

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding. Copyright © 2015 Elsevier GmbH. All rights reserved.

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana)

PubMed Central

Cajimat, Maria N. B.; Milazzo, Mary Louise; Borchert, Jeff N.; Abbott, Ken D.; Bradley, Robert D.; Fulhorst, Charles F.

2008-01-01

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus. PMID:18304671

Correlation between structure, protein composition, morphogenesis and cytopathology of Glossina pallidipes salivary gland hypertrophy virus.

PubMed

Kariithi, Henry M; van Lent, Jan W M; Boeren, Sjef; Abd-Alla, Adly M M; Ince, Ikbal Agah; van Oers, Monique M; Vlak, Just M

2013-01-01

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rod-shaped, enveloped virions. Its 190 kb genome contains 160 putative protein-coding ORFs. Here, the structural components, protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the nucleocapsid, tegument, envelope and helical surface projections, were observed in purified GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within the nuclei of infected salivary gland cells, whereas enveloped virions were located in the cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes disintegrated. Treatment of virions with 1 % NP-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a database search engine, a total of 45 viral proteins were identified. Of these, ten and 15 were associated with the envelope and the nucleocapsid fractions, respectively, whilst 20 were detected in both fractions, most likely representing tegument proteins. In addition, 51 host-derived proteins were identified in the proteome of the virus particle, 13 of which were verified to be incorporated into the mature virion using a proteinase K protection assay. This study provides important information about GpSGHV biology and suggests options for the development of future anti-GpSGHV strategies by interfering with virus-host interactions.

Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

PubMed

Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

2014-07-17

Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. Copyright © 2014 Elsevier B.V. All rights reserved.

Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

PubMed Central

Kolesnikova, Larissa; Welsch, Sonja; Krähling, Verena; Davey, Norman; Parsy, Marie-Laure; Becker, Stephan; Briggs, John A. G.

2011-01-01

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps. PMID:22110401

Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

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

Lee, Eun-Gyung; Linial, Maxine L.

2006-03-30

The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similarmore » to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both {delta}CH1 and {delta}CH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.« less

Construction of yellow fever-influenza A chimeric virus particles.

PubMed

Oliveira, B C E P D; Liberto, M I M; Barth, O M; Cabral, M C

2002-12-01

In order to obtain a better understanding of the functional mechanisms involved in the fusogenesis of enveloped viruses, the influenza A (X31) and the yellow fever (17DD) virus particles were used to construct a chimeric structure based on their distinct pH requirements for fusion, and the distinct malleability of their nucleocapsids. The malleable nucleocapsid of the influenza A virus particle is characterized by a pleomorphic configuration when observed by electron microscopy. A heat inactivated preparation of X31 virus was used as a lectin to interact with the sialic acid domains present in the 17DD virus envelope. The E spikes of 17DD virus were induced to promote fusion of both envelopes, creating a double genome enveloped structure, the chimeric yellow fever-influenza A virus particle. These chimeric viral particles, originally denominated 'partículas virais quiméricas' (PVQ), were characterized by their infectious capacity for different biological systems. Cell inoculation with PVQ resulted in viral products that showed similar characteristics to those obtained after 17DD virus infections. Our findings open new opportunities towards the understanding of both virus particles and aspects of cellular physiologic quality control. The yellow fever-influenza A chimeric particles, by means of their hybrid composition, should be a valuable tool in the study of cell biology and the function of viral components. Copyright 2002 Elsevier Science B.V.

Identification of a common antigenic site in the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

PubMed

Casal, J I; Rodriguez, M J; Sarraseca, J; Garcia, J; Plana-Duran, J; Sanz, A

1998-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein.

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

Nucleotide Sequence of the Hantaan Virus S RNA Segment and Expression of Encoded Proteins

DTIC Science & Technology

1987-11-03

human vaccinia vaccination ). A second dose of virus was given in the same ...vaccinia vector. A necessary first step in vaccine investigation woul d be to determine if animals infected with the two HTV recombinant viruses can ...vaccinia virus (Buller et al., 1985). Mice were infected by tail scarification since it is identical to the method used to vaccinate 169 humans

ICTV virus taxonomy profile: Baculoviridae

USDA-ARS?s Scientific Manuscript database

The Baculoviridae is a family of large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. Virions consist of enveloped rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15 to 15 µm. The occlusion bodies consist of a matrix composed of a single viral...

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

PubMed Central

Füzik, Tibor; Píchalová, Růžena; Schur, Florian K. M.; Strohalmová, Karolína; Křížová, Ivana; Hadravová, Romana; Rumlová, Michaela; Briggs, John A. G.

2016-01-01

ABSTRACT The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This

Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases

PubMed Central

Reguera, Juan; Gerlach, Piotr; Rosenthal, Maria; Gaudon, Stephanie; Coscia, Francesca; Günther, Stephan; Cusack, Stephen

2016-01-01

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase. PMID:27304209

Interaction study of rice stripe virus proteins reveals a region of the nucleocapsid protein (NP) required for NP self-interaction and nuclear localization.

PubMed

Lian, Sen; Cho, Won Kyong; Jo, Yeonhwa; Kim, Sang-Min; Kim, Kook-Hyung

2014-04-01

Rice stripe virus (RSV), which belongs to the genus Tenuivirus, is an emergent virus problem. The RSV genome is composed of four single-strand RNAs (RNA1-RNA4) and encodes seven proteins. We investigated interactions between six of the RSV proteins by yeast-two hybrid (Y2H) assay in vitro and by bimolecular fluorescence complementation (BiFC) in planta. Y2H identified self-interaction of the nucleocapsid protein (NP) and NS3, while BiFC revealed self-interaction of NP, NS3, and NCP. To identify regions(s) and/or crucial amino acid (aa) residues required for NP self-interaction, we generated various truncated and aa substitution mutants. Y2H assay showed that the N-terminal region of NP (aa 1-56) is necessary for NP self-interaction. Further analysis with substitution mutants demonstrated that additional aa residues located at 42-47 affected their interaction with full-length NP. These results indicate that the N-terminal region (aa 1-36 and 42-47) is required for NP self-interaction. BiFC and co-localization studies showed that the region required for NP self-interaction is also required for NP localization at the nucleus. Overall, our results indicate that the N-terminal region (aa 1-47) of the NP is important for NP self-interaction and that six aa residues (42-47) are essential for both NP self-interaction and nuclear localization. Copyright © 2014 Elsevier B.V. All rights reserved.

Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.

PubMed

Ellenbecker, Mary; St Goddard, Jeremy; Sundet, Alec; Lanchy, Jean-Marc; Raiford, Douglas; Lodmell, J Stephen

2015-10-01

Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Herpes simplex virus 1 induces egress channels through marginalized host chromatin

DOE PAGES

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa; ...

2016-06-28

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain

PubMed Central

Bruhn, Jessica F.; Barnett, Katherine C.; Bibby, Jaclyn; Thomas, Jens M. H.; Keegan, Ronan M.; Rigden, Daniel J.; Bornholdt, Zachary A.

2014-01-01

The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle. PMID:24155387

Characterization of a single-nucleocapsid nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia.

PubMed

Cheng, X W; Carner, G R

2000-05-01

A single-nucleocapsid nucleopolyhedrovirus (NPV) isolated from Thysanoplusia orichalcea L. (Lepidoptera:Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1. 22 microm (range = 0.803-1.931 microm). The length of the virion with an envelope averaged 0.29 and 0.23 microm without an envelope. ThorNPV was propagated in Pseudoplusia includens (Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV produced in T. orichalcea and P. includens. Polyhedrin amino acid sequence analysis revealed that ThorNPV belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV against third instar P. includens was low, with a LD(50) value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions embedded, which might explain the low virulence against P. includens. Differences in virion occlusion rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD(50) value to P. includens against Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens, P. includens was the only species infected. The virus infected primarily the fat body, tracheal epithelium, and hypodermis. The genomic size of the ThorNPV is 135 kb. Copyright 2000 Academic Press.

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

Dengue Virus Genome Uncoating Requires Ubiquitination.

PubMed

Byk, Laura A; Iglesias, Néstor G; De Maio, Federico A; Gebhard, Leopoldo G; Rossi, Mario; Gamarnik, Andrea V

2016-06-28

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade

Nucleocapsid Gene-Mediated Transgenic Resistance Provides Protection Against Tomato spotted wilt virus Epidemics in the Field.

PubMed

Herrero, S; Culbreath, A K; Csinos, A S; Pappu, H R; Rufty, R C; Daub, M E

2000-02-01

ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.

Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

PubMed

Baker, Laura E; Ellena, Jeffrey F; Handing, Katarzyna B; Derewenda, Urszula; Utepbergenov, Darkhan; Engel, Daniel A; Derewenda, Zygmunt S

2016-01-01

The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV). In each of these viruses the ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. It is tightly associated with the viral RNA in the nucleocapsid, and during the lifecycle of the virus is essential for transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. The structure of the unique C-terminal globular domain of the NP from EBOV has recently been determined and shown to be structurally unrelated to any other known protein [Dziubańska et al. (2014), Acta Cryst. D70, 2420-2429]. In this paper, a study of the C-terminal domains from the NP from the remaining four species of Ebolavirus, as well as from the MARV strain of Marburgvirus, is reported. As expected, the crystal structures of the BDBV and TAFV proteins show high structural similarity to that from EBOV, while the MARV protein behaves like a molten globule with a core residual structure that is significantly different from that of the EBOV protein.

A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus▿†

PubMed Central

Jones, Daniel M.; Atoom, Ali M.; Zhang, Xiaozhen; Kottilil, Shyamasundaran; Russell, Rodney S.

2011-01-01

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1T-64–66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1T-64–66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1T-64–66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly. PMID:21957313

A Thermodynamic Model for Genome Packaging in Hepatitis B Virus.

PubMed

Kim, Jehoon; Wu, Jianzhong

2015-10-20

Understanding the fundamentals of genome packaging in viral capsids is important for finding effective antiviral strategies and for utilizing benign viral particles for gene therapy. While the structure of encapsidated genomic materials has been routinely characterized with experimental techniques such as cryo-electron microscopy and x-ray diffraction, much less is known about the molecular driving forces underlying genome assembly in an intracellular environment and its in vivo interactions with the capsid proteins. Here we study the thermodynamic basis of the pregenomic RNA encapsidation in human Hepatitis B virus in vivo using a coarse-grained molecular model that captures the essential components of nonspecific intermolecular interactions. The thermodynamic model is used to examine how the electrostatic interaction between the packaged RNA and the highly charged C-terminal domains (CTD) of capsid proteins regulate the nucleocapsid formation. The theoretical model predicts optimal RNA content in Hepatitis B virus nucleocapsids with different CTD lengths in good agreement with mutagenesis measurements, confirming the predominant role of electrostatic interactions and molecular excluded-volume effects in genome packaging. We find that the amount of encapsidated RNA is not linearly correlated with the net charge of CTD tails as suggested by earlier theoretical studies. Our thermodynamic analysis of the nucleocapsid structure and stability indicates that ∼10% of the CTD residues are free from complexation with RNA, resulting in partially exposed CTD tails. The thermodynamic model also predicts the free energy of complex formation between macromolecules, which corroborates experimental results for the impact of CTD truncation on the nucleocapsid stability. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus

PubMed Central

Yabukarski, Filip; Blocquel, David; Schneider, Robert; Tarbouriech, Nicolas; Papageorgiou, Nicolas; Ruigrok, Rob W. H.; Jamin, Marc; Jensen, Malene Ringkjøbing; Longhi, Sonia; Blackledge, Martin

2013-01-01

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, NTAIL, of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered NTAIL domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between NTAIL and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of NTAIL upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to NTAIL without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae. PMID:24086133

Atomic resolution description of the interaction between the nucleoprotein and phosphoprotein of Hendra virus.

PubMed

Communie, Guillaume; Habchi, Johnny; Yabukarski, Filip; Blocquel, David; Schneider, Robert; Tarbouriech, Nicolas; Papageorgiou, Nicolas; Ruigrok, Rob W H; Jamin, Marc; Jensen, Malene Ringkjøbing; Longhi, Sonia; Blackledge, Martin

2013-01-01

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, N(TAIL), of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N(TAIL) domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N(TAIL) and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of N(TAIL) upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to N(TAIL) without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.

Epizootiology of Tacaribe serocomplex viruses (Arenaviridae) associated with neotomine rodents (Cricetidae, Neotominae) in southern California.

PubMed

Milazzo, Mary Louise; Cajimat, Maria N B; Mauldin, Matthew R; Bennett, Stephen G; Hess, Barry D; Rood, Michael P; Conlan, Christopher A; Nguyen, Kiet; Wekesa, J Wakoli; Ramos, Ronald D; Bradley, Robert D; Fulhorst, Charles F

2015-02-01

The objective of this study was to advance our knowledge of the epizootiology of Bear Canyon virus and other Tacaribe serocomplex viruses (Arenaviridae) associated with wild rodents in California. Antibody (immunoglobulin G [IgG]) to a Tacaribe serocomplex virus was found in 145 (3.6%) of 3977 neotomine rodents (Cricetidae: Neotominae) captured in six counties in southern California. The majority (122 or 84.1%) of the 145 antibody-positive rodents were big-eared woodrats (Neotoma macrotis) or California mice (Peromyscus californicus). The 23 other antibody-positive rodents included a white-throated woodrat (N. albigula), desert woodrat (N. lepida), Bryant's woodrats (N. bryanti), brush mice (P. boylii), cactus mice (P. eremicus), and deer mice (P. maniculatus). Analyses of viral nucleocapsid protein gene sequence data indicated that Bear Canyon virus is associated with N. macrotis and/or P. californicus in Santa Barbara County, Los Angeles County, Orange County, and western Riverside County. Together, analyses of field data and antibody prevalence data indicated that N. macrotis is the principal host of Bear Canyon virus. Last, the analyses of viral nucleocapsid protein gene sequence data suggested that the Tacaribe serocomplex virus associated with N. albigula and N. lepida in eastern Riverside County represents a novel species (tentatively named "Palo Verde virus") in the genus Arenavirus.

Patchwork structure-function analysis of the Sendai virus matrix protein.

PubMed

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

PubMed

Rodriguez, M J; Sarraseca, J; Garcia, J; Sanz, A; Plana-Durán, J; Ignacio Casal, J

1997-09-01

Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50-66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus*

PubMed Central

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-01-01

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (NTAIL). XD binding induces NTAIL α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced NTAIL folding, XD-NTAIL binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced NTAIL α-helical folding were created within Box-2 of Edmonston MeV NTAIL. Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-NTAIL binding affinity or reduction/loss of XD-induced NTAIL alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity. PMID:22318731

Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.

PubMed

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-04-06

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.

Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein 7.

PubMed

Saha, Mrinmoy; Scerba, Michael T; Shank, Nathaniel I; Hartman, Tracy L; Buchholz, Caitlin A; Buckheit, Robert W; Durell, Stewart R; Appella, Daniel H

2017-05-22

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC 50 values ranging from 1 to 100 μm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure-activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

PubMed

Joseph, Narcisse Ms; Ho, Kok Lian; Tey, Beng Ti; Tan, Chon Seng; Shafee, Norazizah; Tan, Wen Siang

2016-07-08

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016. © 2016 American Institute of Chemical Engineers.

Electron cryo-microscopy structure of Ebola nucleoprotein reveals a mechanism for nucleocapsid-like assembly

PubMed Central

Su, Zhaoming; Wu, Chao; Shi, Liuqing; Luthra, Priya; Pintilie, Grigore D.; Johnson, Britney; Porter, Justin R.; Ge, Peng; Chen, Muyuan; Liu, Gai; Frederick, Thomas E.; Binning, Jennifer M.; Bowman, Gregory R.; Zhou, Z. Hong; Basler, Christopher F.; Gross, Michael L.; Leung, Daisy W.

2018-01-01

Summary Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22–α23. Biochemical, biophysical, and mutational analysis revealed inter-eNP contacts within α22–α23 are critical for viral NC-assembly and regulate viral RNA synthesis. These observations suggest that the N-terminus and α22–α23 of eNP function as context dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target. PMID:29474922

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

PubMed

Thomas, D; Newcomb, W W; Brown, J C; Wall, J S; Hainfeld, J F; Trus, B L; Steven, A C

1985-05-01

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

PubMed Central

Thomas, D; Newcomb, W W; Brown, J C; Wall, J S; Hainfeld, J F; Trus, B L; Steven, A C

1985-01-01

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle.

PubMed

Biedenkopf, Nadine; Lier, Clemens; Becker, Stephan

2016-05-15

Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

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

Huang Jialing, E-mail: hjialing@mail.med.upenn.edu; Lazear, Helen M., E-mail: Hlazear@DOM.wustl.edu; Friedman, Harvey M., E-mail: hfriedma@mail.med.upenn.ed

2011-01-05

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infectedmore » with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.« less

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

PubMed

Thakkar, Vidhi D; Cox, Robert M; Sawatsky, Bevan; da Fontoura Budaszewski, Renata; Sourimant, Julien; Wabbel, Katrin; Makhsous, Negar; Greninger, Alexander L; von Messling, Veronika; Plemper, Richard K

2018-04-15

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral

ICTV Virus Taxonomy Profile: Ophioviridae

PubMed Central

Bó, Elena Dal; da Graça, John V.; Gago-Zachert, Selma; Hammond, John; Moreno, Pedro; Natsuaki, Tomohide; Pallás, Vicente; Navarro, Jose A.; Reyes, Carina A.; Luna, Gabriel Robles; Sasaya, Takahide; Tzanetakis, Ioannis E.; Vaira, Anna María; Verbeek, Martin

2017-01-01

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae. PMID:28635587

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

PubMed

De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

1993-02-25

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

PubMed Central

De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

1993-01-01

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger. Images PMID:8451185

Osteoclast Inhibitory Peptide-1 Therapy for Paget’s Disease

DTIC Science & Technology

2012-08-01

1 (SQSTM1/p62) gene have been widely identified in PDB patients. We previously detected expression of measles virus nucleocapsid (MVNP) transcripts...high bone turnover in PDB. 15. SUBJECT TERMS Paget’s Disease, measles virus nucleocapsid, sequestosome1 , osteoclast, osteoclast inhibitory peptide...detected expression of measles virus nucleocapsid (MVNP) transcripts in osteoclasts from patients with PDB. Also, we have shown that MVNP gene

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

PubMed

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Isolation of Ganjam virus from ticks collected off domestic animals around Pune, Maharashtra, India.

PubMed

Joshi, M V; Geevarghese, G; Joshi, G D; Ghodke, Y S; Mourya, D T; Mishra, A C

2005-03-01

Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.

Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation.

PubMed

Yang, Li; Wang, Ya-Juan; Chen, Hai-Jun; Shi, Li-Ping; Tong, Xian-Kun; Zhang, Yang-Ming; Wang, Gui-Feng; Wang, Wen-Long; Feng, Chun-Lan; He, Pei-Lan; Xu, Yi-Bin; Lu, Meng-Ji; Tang, Wei; Nan, Fa-Jun; Zuo, Jian-Ping

2016-01-01

During the hepatitis B virus (HBV) life cycle, nucleocapsid assembly is essential for HBV replication. Both RNA reverse transcription and DNA replication occur within the HBV nucleocapsid. HBV nucleocapsid is consisted of core protein (HBcAg), whose carboxy-terminal domain (CTD) contains an Arg-rich domain (ARD). The ARD of HBcAg does contribute to the encapsidation of pregenomic RNA (pgRNA). Previously, we reported a small-molecule, NZ-4, which dramatically reduced the HBV DNA level in an in vitro cell setting. Here, we explore the possible mechanisms by which NZ-4 inhibits HBV function. As an HBV inhibitor, NZ-4 leads to the formation of genome-free capsids, including a new population of capsid that runs faster on agarose gels. NZ-4's activity was dependent on the presence of the ARD I, containing at least one positively charged amino acid. NZ-4 might provide a new option for further development of HBV therapeutics for the treatment of chronic hepatitis B. Copyright © 2015. Published by Elsevier B.V.

Characterization of Nipah Virus from Naturally Infected Pteropus vampyrus Bats, Malaysia

PubMed Central

Hassan, Sharifah S.; Olival, Kevin J.; Mohamed, Maizan; Chang, Li-Yen; Hassan, Latiffah; Saad, Norsharina M.; Shohaimi, Syamsiah A.; Mamat, Zaini C.; Naim, M.S.; Epstein, Jonathan H.; Suri, Arshad S.; Field, Hume E.; Daszak, Peter

2010-01-01

We isolated and characterized Nipah virus (NiV) from Pteropus vampyrus bats, the putative reservoir for the 1998 outbreak in Malaysia, and provide evidence of viral recrudescence. This isolate is monophyletic with previous NiVs in combined analysis, and the nucleocapsid gene phylogeny suggests that similar strains of NiV are co-circulating in sympatric reservoir species. PMID:21122240

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

PubMed

De, B P; Galinski, M S; Banerjee, A K

1990-03-01

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

A necrotizing pneumonia in lambs caused by pseudorabies virus (Aujesky's disease virus).

PubMed Central

Schmidt, S P; Pirtle, E C; Hagemoser, W A; Wages, D P

1987-01-01

An outbreak of pseudorabies occurred in sheep housed with swine in the same building. Although the sheep and swine were not in physical contact, the lambs and ewes were exposed to air from the sows' section. Three dead lambs were submitted to the Iowa State University Veterinary Diagnostic Laboratory for necropsy. Grossly there were pulmonary congestion and multifocal pulmonary hemorrhages. Microscopic lesions were severe acute multifocal necrotizing bronchopneumonia with necrotizing vasculitis and intranuclear inclusion bodies within the neurons of the parabronchial ganglia. Bacterial cultures were negative for pathogenic agents; pseudorabies virus was isolated from ovine brain tissue. Viral antigen was demonstrated in the neurons of the parabronchial ganglia by immunoperoxidase staining. Electron microscopy revealed nucleocapsids in the parabronchial ganglionic neurons which contained basophilic intranuclear inclusion bodies. Viral DNA prepared from the ovine pseudorabies virus isolate was found by restriction endonuclease analysis to be related to the Indiana Funkhauser strain of pseudorabies virus. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3032388

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed

Jin, H; Elliott, R M

1993-03-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed Central

Jin, H; Elliott, R M

1993-01-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities. Images PMID:8437222

Autographa californica Multiple Nucleopolyhedrovirus ac75 Is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation.

PubMed

Shi, Anqi; Hu, Zhaoyang; Zuo, Yachao; Wang, Yan; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2018-02-15

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 ( ac75 ) is a highly conserved gene of unknown function. In this study, we constructed an ac75 knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms, of 18 and 15 kDa, of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BVs but with only the nucleocapsid fraction of ODVs; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was localized predominantly in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm and at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identified ac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. IMPORTANCE During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are

Recombinant Marburg Virus Expressing EGFP Allows Rapid Screening of Virus Growth and Real-time Visualization of Virus Spread

PubMed Central

Schmidt, Kristina Maria; Schümann, Michael; Olejnik, Judith; Krähling, Verena

2011-01-01

The generation of recombinant enhanced green fluorescent protein (EGFP)--expressing viruses has significantly improved the study of their life cycle and opened up the possibility for the rapid screening of antiviral drugs. Here we report rescue of a recombinant Marburg virus (MARV) expressing EGFP from an additional transcription unit (ATU). The ATU was inserted between the second and third genes, encoding VP35 and VP40, respectively. Live-cell imaging was used to follow virus spread in real time. EGFP expression was detected at 32 hours postinfection (hpi), and infection of neighboring cells was monitored at 55 hpi. Compared to the parental virus, production of progeny rMARV-EGFP was reduced 4-fold and lower protein levels of VP40, but not nucleoprotein, were observed, indicating a decrease in downstream protein expression due to the insertion of an ATU. Interestingly, EGFP concentrated in viral inclusions in infected cells. This was reproduced by transient expression of both EGFP and other fluorescent proteins along with filovirus nucleocapsid proteins, and may suggest that a general increase in protein synthesis occurs at viral inclusion sites. In conclusion, the EGFP-expressing MARV will be a useful tool not only to monitor virus spread and screen for antiviral compounds, but also to investigate the biology of inclusion body formation. PMID:21987762

Case report: epithelial intracytoplasmic herpes viral inclusions associated with an outbreak of duck virus enteritis

USGS Publications Warehouse

Barr, B.C.; Jessup, David A.; Docherty, Douglas E.; Lownestine, L.J.

1992-01-01

Several muscovy ducks from a free-roaming flock of 65 muscovy and mallard ducks died over a 3-week period. Three muscovy ducks were necropsied. Gross and microscopic changes were compatible with duck virus enteritis, and the virus was isolated. In addition to intranuclear viral inclusion bodies in several tissues, intracytoplasmic inclusion bodies were present in esophageal and cloacal epithelium, By electron microscopy, the membrane-bound intracytoplasmic inclusions were found to contain enveloped herpesvirus, and nuclei contained herpes viral nucleocapsids.

Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates.

PubMed

Stanković, I; Bulajić, A; Vučurović, A; Ristić, D; Milojević, K; Berenji, J; Krstić, B

2011-01-01

In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most frequently found virus at the localities of Central Serbia, while PVY and CMV were the most frequent viruses in the Vojvodina Province. Single infections were prevalent in years 2005-2007 and the most frequent were those of PVY. A triple combination of those viruses was most frequent mixed infection type in 2008. The presence of all five detected viruses was confirmed in selected ELISA-positive samples by RT-PCR and sequencing. The comparisons of obtained virus isolate sequences with those available in NCBI, confirmed the authenticity of serologically detected viruses. Phylogenetic analysis based on partial nucleocapsid gene sequences revealed a joint clustering of Serbian, Bulgarian and Montenegrin TSWV isolates into one geographic subpopulation, which was distinct from the other subpopulation of TSWV isolates from the rest of the European countries. The high incidence of viruses in Serbian tobacco crops highlights the importance of enhancing farmers knowledge towards better implementation of control strategies for preventing serious losses.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

PubMed

Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

2016-06-01

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay To Detect Antibodies to Viral Hemorrhagic Septicemia Virus

PubMed Central

Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas

2014-01-01

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV. PMID:24429071

Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

USGS Publications Warehouse

Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

2014-01-01

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.

PubMed

Bloyet, Louis-Marie; Brunel, Joanna; Dosnon, Marion; Hamon, Véronique; Erales, Jenny; Gruet, Antoine; Lazert, Carine; Bignon, Christophe; Roche, Philippe; Longhi, Sonia; Gerlier, Denis

2016-12-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

PubMed Central

Hamon, Véronique; Erales, Jenny; Bignon, Christophe; Roche, Philippe

2016-01-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region. PMID:27936158

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

Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.

PubMed

Das, Subash C; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K

2009-03-01

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-DeltaM-Mtc and VSV-DeltaM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-DeltaM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-DeltaM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

Screening and structure-based modeling of T-cell epitopes of Nipah virus proteome: an immunoinformatic approach for designing peptide-based vaccine.

PubMed

Kamthania, Mohit; Sharma, D K

2015-12-01

Identification of Nipah virus (NiV) T-cell-specific antigen is urgently needed for appropriate diagnostic and vaccination. In the present study, prediction and modeling of T-cell epitopes of Nipah virus antigenic proteins nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, L protein, W protein, V protein and C protein followed by the binding simulation studies of predicted highest binding scorers with their corresponding MHC class I alleles were done. Immunoinformatic tool ProPred1 was used to predict the promiscuous MHC class I epitopes of viral antigenic proteins. The molecular modelings of the epitopes were done by PEPstr server. And alleles structure were predicted by MODELLER 9.10. Molecular dynamics (MD) simulation studies were performed through the NAMD graphical user interface embedded in visual molecular dynamics. Epitopes VPATNSPEL, NPTAVPFTL and LLFVFGPNL of Nucleocapsid, V protein and Fusion protein have considerable binding energy and score with HLA-B7, HLA-B*2705 and HLA-A2MHC class I allele, respectively. These three predicted peptides are highly potential to induce T-cell-mediated immune response and are expected to be useful in designing epitope-based vaccines against Nipah virus after further testing by wet laboratory studies.

Hydrophobic Amino Acids in the Human Immunodeficiency Virus Type 1 p2 and Nucleocapsid Proteins Can Contribute to the Rescue of Deleted Viral RNA Packaging Signals

PubMed Central

Rong, Liwei; Russell, Rodney S.; Hu, Jing; Guan, Yongjun; Kleiman, Lawrence; Liang, Chen; Wainberg, Mark A.

2001-01-01

An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5′ major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a cis manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses. PMID:11461996

Detection of Nipah virus RNA in fruit bat (Pteropus giganteus) from India.

PubMed

Yadav, Pragya D; Raut, Chandrashekhar G; Shete, Anita M; Mishra, Akhilesh C; Towner, Jonathan S; Nichol, Stuart T; Mourya, Devendra T

2012-09-01

The study deals with the survey of different bat populations (Pteropus giganteus, Cynopterus sphinx, and Megaderma lyra) in India for highly pathogenic Nipah virus (NiV), Reston Ebola virus, and Marburg virus. Bats (n = 140) from two states in India (Maharashtra and West Bengal) were tested for IgG (serum samples) against these viruses and for virus RNAs. Only NiV RNA was detected in a liver homogenate of P. giganteus captured in Myanaguri, West Bengal. Partial sequence analysis of nucleocapsid, glycoprotein, fusion, and phosphoprotein genes showed similarity with the NiV sequences from earlier outbreaks in India. A serum sample of this bat was also positive by enzyme-linked immunosorbent assay for NiV-specific IgG. This is the first report on confirmation of Nipah viral RNA in Pteropus bat from India and suggests the possible role of this species in transmission of NiV in India.

Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus

USDA-ARS?s Scientific Manuscript database

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12,792 nucleotides long and organized into seven open reading frames with the gene order 3’-N-X-P-Y-M-G-L-5’, which encodes the nucleocapsid, phosphoprotein, movement, matrix, glycoprotein and RNA-d...

Cryo-Electron Tomography of Rubella Virus

PubMed Central

Battisti, Anthony J.; Yoder, Joshua D.; Plevka, Pavel; Winkler, Dennis C.; Mangala Prasad, Vidya; Kuhn, Richard J.; Frey, Teryl K.; Steven, Alasdair C.

2012-01-01

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses. PMID:22855483

Cryo-electron tomography of rubella virus.

PubMed

Battisti, Anthony J; Yoder, Joshua D; Plevka, Pavel; Winkler, Dennis C; Prasad, Vidya Mangala; Kuhn, Richard J; Frey, Teryl K; Steven, Alasdair C; Rossmann, Michael G

2012-10-01

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

PubMed

Watt, Ari; Moukambi, Felicien; Banadyga, Logan; Groseth, Allison; Callison, Julie; Herwig, Astrid; Ebihara, Hideki; Feldmann, Heinz; Hoenen, Thomas

2014-09-01

Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study

A Retrospective Analysis of Sera Collected by the Hemorrhagic Fever Commission during the Korean Conflict

DTIC Science & Technology

1990-05-01

of leptospirosis ; no diagnosis could be made for the other 14. virus-specific pooled mouse monoclonal antibodies or control fluids: Seropositive...day 7 of disease. Further, 1184 Concise Communications JID 1990:162 INovember) 100,00o - tion of a single case of leptospirosis , the etiology of the...prototype Hantaan tibodies to Rift Valley fever virus in ovine and bovine sera. Am JVet Res 1987:48:1138-1141virus as the causative agent of hemorrhagic

Transmission of haemorrhagic fever with renal syndrome in china and the role of climate factors: a review.

PubMed

Hansen, Alana; Cameron, Scott; Liu, Qiyong; Sun, Yehuan; Weinstein, Philip; Williams, Craig; Han, Gil-Soo; Bi, Peng

2015-04-01

Haemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease that poses a serious public health threat in China. HFRS is caused by hantaviruses, mainly Seoul virus in urban areas and Hantaan virus in agricultural areas. Although preventive measures including vaccination programs and rodent control measures have resulted in a decline in cases in recent years, there has been an increase in incidence in some areas and new endemic areas have emerged. This review summarises the recent literature relating to the effects of climatic factors on the incidence of HFRS in China and discusses future research directions. Temperature, precipitation and humidity affect crop yields, rodent breeding patterns and disease transmission, and these can be influenced by a changing climate. Detailed surveillance of infections caused by Hantaan and Seoul viruses and further research on the viral agents will aid in interpretation of spatiotemporal patterns and a better understanding of the environmental and ecological drivers of HFRS amid China's rapidly urbanising landscape and changing climate. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Microscopic Characterization of the Brazilian Giant Samba Virus.

PubMed

Schrad, Jason R; Young, Eric J; Abrahão, Jônatas S; Cortines, Juliana R; Parent, Kristin N

2017-02-14

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses .

Microscopic Characterization of the Brazilian Giant Samba Virus

PubMed Central

Schrad, Jason R.; Young, Eric J.; Abrahão, Jônatas S.; Cortines, Juliana R.; Parent, Kristin N.

2017-01-01

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses. PMID:28216551

SLAM- and nectin-4-independent noncytolytic spread of canine distemper virus in astrocytes.

PubMed

Alves, Lisa; Khosravi, Mojtaba; Avila, Mislay; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Vandevelde, Marc; Plattet, Philippe

2015-05-01

Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered "nectin-4-blind" recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS) infections is that

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus

PubMed Central

Mangala Prasad, Vidya

2017-01-01

Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway. PMID:28575072

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus.

PubMed

Mangala Prasad, Vidya; Klose, Thomas; Rossmann, Michael G

2017-06-01

Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.

[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.

Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods.

PubMed

Khandogin, Jana; Musier-Forsyth, Karin; York, Darrin M

2003-07-25

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) plays several important roles in the viral life-cycle and presents an attractive target for rational drug design. Here, the macromolecular reactivity of NC and its binding to RNA is characterized through determination of electrostatic and chemical descriptors derived from linear-scaling quantum calculations in solution. The computational results offer a rationale for the experimentally observed susceptibility of the Cys49 thiolate toward small-molecule electrophilic agents, and support the recently proposed stepwise protonation mechanism of the C-terminal Zn-coordination complex. The distinctive binding mode of NC to SL2 and SL3 stem-loops of the HIV-1 genomic RNA packaging signal is studied on the basis of protein side-chain contributions to the electrostatic binding energies. These results indicate the importance of several basic residues in the 3(10) helical region and the N-terminal zinc finger, and rationalize the presence of several evolutionarily conserved residues in NC. The combined reactivity and RNA-binding study provides new insights that may contribute toward the structure-based design of anti-HIV therapies.

Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

PubMed

Jackson, D P; Percy, D H; Morris, V L

1984-09-01

When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed

Tweeten, K A; Bulla, L A; Consigli, R A

1977-09-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Tomato Spotted Wilt Virus Particle Morphogenesis in Plant Cells

PubMed Central

Kikkert, Marjolein; Van Lent, Jan; Storms, Marc; Bodegom, Pentcho; Kormelink, Richard; Goldbach, Rob

1999-01-01

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed. PMID:9971812

The first phlebo-like virus infecting plants: a case study on the adaptation of negative-stranded RNA viruses to new hosts.

PubMed

Navarro, Beatriz; Minutolo, Maria; De Stradis, Angelo; Palmisano, Francesco; Alioto, Daniela; Di Serio, Francesco

2018-05-01

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

PubMed

Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

2016-06-03

Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biology and genomics of viruses within the genus Gammabaculovirus.

PubMed

Arif, Basil; Escasa, Shannon; Pavlik, Lillian

2011-11-01

Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

PubMed

Truong, A T; Mulders, M N; Gautam, D C; Ammerlaan, W; de Swart, R L; King, C C; Osterhaus, A D; Muller, C P

2001-07-01

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Development and Characterization of an Infectious cDNA Clone of the Modified Live Virus Vaccine Strain of Equine Arteritis Virus

PubMed Central

Zhang, Jianqiang; Go, Yun Young; Huang, Chengjin M.; Meade, Barry J.; Lu, Zhengchun; Snijder, Eric J.; Timoney, Peter J.

2012-01-01

A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals. PMID:22739697

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

PubMed Central

Rice, W G; Schaeffer, C A; Graham, L; Bu, M; McDougal, J S; Orloff, S L; Villinger, F; Young, M; Oroszlan, S; Fesen, M R

1993-01-01

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle. Images Fig. 3 Fig. 4 PMID:7692451

Hemorrhagic fever with renal syndrome accompanied by panhypopituitarism and central diabetes insipidus: a case report.

PubMed

Ahn, Hee Jung; Chung, Jong-Hoon; Kim, Dong-Min; Yoon, Na-Ra; Kim, Choon-Mee

2018-03-05

Central diabetes insipidus (DI) was detected in a patient with hemorrhagic fever with renal syndrome (HFRS) who had been molecularly and serologically diagnosed with Hantaan virus infection. We recommend that clinicians differentiate central DI in HFRS patients with a persistent diuretic phase even when pituitary MRI findings are normal.

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

Dengue Virus Genome Uncoating Requires Ubiquitination

PubMed Central

Byk, Laura A.; Iglesias, Néstor G.; De Maio, Federico A.; Gebhard, Leopoldo G.; Rossi, Mario

2016-01-01

ABSTRACT The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. PMID:27353759

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed Central

Tweeten, K A; Bulla, L A; Consigli, R A

1977-01-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid. Images PMID:334076

Actin filaments participate in West Nile (Sarafend) virus maturation process.

PubMed

Chu, J J H; Choo, B G H; Lee, J W M; Ng, M L

2003-11-01

West Nile (Sarafend) virus has previously been shown to egress by budding at the plasma membrane of infected cells, but relatively little is known about the mechanism involved in this mode of release. During the course of this study, it was discovered that actin filaments take part in the virus maturation process. Using dual-labeled immunofluorescence and immunoelectron microscopy at late infection (10 hr p.i.), co-localization of viral structural (envelope and capsid) proteins with actin filaments was confirmed. The virus structural proteins were also immunoprecipitated with anti-actin antibody, further demonstrating the strong association between the two components. Perturbation of actin filaments by cytochalasin B strongly inhibited the release of West Nile virus (approximately 10,000-fold inhibition) when compared with the untreated cells. Infectious virus particles were recovered after the removal of cytochalasin B. Further confirmation was obtained when nucleocapsid particles were found associated with disrupted actin filaments at the periphery of cytochalasin B-treated cells. Together, these results showed that actin filaments do indeed have a key role in the release of West Nile (Sarafend) virions. Copyright 2003 Wiley-Liss, Inc.

Molecular characterization of Nipah virus from Pteropus hypomelanus in Southern Thailand.

PubMed

Wacharapluesadee, Supaporn; Samseeneam, Panumas; Phermpool, Mana; Kaewpom, Thongchai; Rodpan, Apaporn; Maneeorn, Pattarapol; Srongmongkol, Phimchanok; Kanchanasaka, Budsabong; Hemachudha, Thiravat

2016-03-25

Nipah virus (NiV) first emerged in Malaysia in 1998, with two bat species (Pteropus hypomelanus and P. vampyrus) as the putative natural reservoirs. In 2002, NiV IgG antibodies were detected in these species from Thailand, but viral RNA could not be detected for strain characterization. Two strains of NiV (Malaysia and Bangladesh) have been found in P. lylei in central Thailand, although Bangladesh strain, the causative strain for the outbreak in Bangladesh since 2001, was dominant. To understand the diversity of NiV in Thailand, this study identified NiV strain, using molecular characterizations, from P. hypomelanus in southern Thailand. Pooled bat urine specimens were collected from plastic sheet underneath bat roosts in April 2010, and then monthly from December 2010 to May 2011 at an island in southern Thailand. Five in 184 specimens were positive for NiV, using duplex nested RT-PCR assay on partial nucleocapsid fragment (357 bp). Whole sequences of nucleocapsid gene from four bats were characterized. All 5 partial fragments and 4 whole nucleocapsid genes formed a monophyletic with NiV-MY. Our study showed that P. hypomelanus in southern Thailand and from Malaysia, a bordering country, harbored similar NiV. This finding indicates that NiV is not limited to central Thailand or P. lylei species, and it may be a source of inter-species transmission. This indicates a higher potential for a widespread NiV outbreak in Thailand. NiV surveillance in Pteropus bats, the major natural reservoirs, should be conducted continuously in countries or regions with high susceptibility to outbreaks.

Subcellular distribution and life cycle of Epstein-Barr virus in keratinocytes of oral hairy leukoplakia.

PubMed Central

Rabanus, J. P.; Greenspan, D.; Petersen, V.; Leser, U.; Wolf, H.; Greenspan, J. S.

1991-01-01

The authors investigated the life cycle of Epstein-Barr virus (EBV) in keratinocytes of oral hairy leukoplakia by combining immunohistochemistry. DNA in situ hybridization, and lectin histochemistry with electron microscopy. Diffuse-staining components of the EBV early antigen complex (EA-D), EBV 150-kd capsid antigen (VCA), EBV membrane antigen (gp350/220), and double-stranded DNA were labeled with monoclonal antibodies. An EBV-DNA probe was used to locate EBV DNA. Wheat-germ agglutinin (WGA) was employed to distinguish Golgi-associated compartments. The authors found EBV proteins and EBV DNA only in keratinocytes with apparent viral assembly. In situ hybridization showed EBV DNA in free corelike material and in electron-dense cores of mature nucleocapsids. Monoclonal antibodies to nonspecific double-stranded DNA attached to the same structures and to marginated chromatin. Components of EA-D were dispersed throughout the nuclei but accumulated near condensed chromatin and in 'punched-out' regions of the chromatin. Epstein-Barr virus 150-kd capsid antigen was found only in the nuclei, where it appeared preferentially on mature nucleocapsids. As yet unexplained arrays of intranuclear particles that remained unlabeled with all EBV-specific probes reacted intensely with an antiserum against common papillomavirus antigen. Gp350/220 was detectable in various cellular membrane compartments and was highly concentrated on EBV envelopes in peripheral Golgi-associated secretory vesicles. It was less abundant on the extracellular EBV, indicating that viral membrane antigen partly dissociates from the mature virus. Combined lectin-binding histochemistry and electron microscopy demonstrated for the first time that EBV is processed in the Golgi apparatus, which eventually releases the virus by fusion with the plasma membrane. These results provide insight into the biologic events that occur during complete EBV replication in vivo. Images Figure 1 Figure 2 Figure 3 Figure 4

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid.

PubMed

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D; Klose, Thomas; Chipman, Paul R; Van Etten, James L; Rossmann, Michael G

2011-09-06

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid

PubMed Central

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D.; Klose, Thomas; Chipman, Paul R.; Van Etten, James L.; Rossmann, Michael G.

2011-01-01

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions. PMID:21873222

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

PubMed

Liu, Jie; Ochieng, Caroline; Wiersma, Steve; Ströher, Ute; Towner, Jonathan S; Whitmer, Shannon; Nichol, Stuart T; Moore, Christopher C; Kersh, Gilbert J; Kato, Cecilia; Sexton, Christopher; Petersen, Jeannine; Massung, Robert; Hercik, Christine; Crump, John A; Kibiki, Gibson; Maro, Athanasia; Mujaga, Buliga; Gratz, Jean; Jacob, Shevin T; Banura, Patrick; Scheld, W Michael; Juma, Bonventure; Onyango, Clayton O; Montgomery, Joel M; Houpt, Eric; Fields, Barry

2016-01-01

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Temperature-sensitive mutants of measles virus produced from persistently infected HeLa cells.

PubMed

Armen, R C; Evermann, J F; Truant, A L; Laughlin, C A; Hallum, J V

1977-01-01

A persistent infection with the Edmonston strain of measles virus was established in HeLa cells in the absence of measles virus antibody (HeLaPI cells). By hemadsorption or immunofluoresnce virtually 100 per cent of the cells possessed measles virus components. HeLaPI cells produced no interferon and were not resistant to superinfection with Newcastle disease virus. HeLaPI cells contained both smooth (15--18 nm) and rought (20--35 nm) nucleocapsids as detected by electron microscopy. The virus produced from the HeLaPI cells (MVPI) varied in titer between 1.5 X 10(2) and 5.5 X10(4) PFU/ml, had a smaller plque size and was more heat resistant than wild-type measles virus. MVPI was also found to be temperature-sensitive. The temperature-sensitivity of MVPI was determined by the efficiency of plaquing at 33 degrees and 39 degrees C in Vero cell monolayers. When HeLaPI cells were incubated at 33 degrees C, there was a 50-fold increase in virus production as well as a slight increase in the percentage of cells forming infectious centers compared to HeLaPI cells grown at 37 degrees C. MVPI readily established a persistent infection in HeLa cells which also rleased temperature-sensitive virus.

Successful topical respiratory tract immunization of primates against Ebola virus.

PubMed

Bukreyev, Alexander; Rollin, Pierre E; Tate, Mallory K; Yang, Lijuan; Zaki, Sherif R; Shieh, Wun-Ju; Murphy, Brian R; Collins, Peter L; Sanchez, Anthony

2007-06-01

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

Detection of infectious bronchitis virus strains similar to Japan in Taiwan.

PubMed

Tsai, Cheng-Ta; Tsai, Hsin-Fu; Wang, Ching-Ho

2016-06-01

A total of 1,320 tracheal samples from 66 broiler flocks sent to slaughterhouses and 42 tracheal samples from 42 flocks of local chickens in the field were collected for infectious bronchitis virus (IBV) gene detection by reverse transcription polymerase chain reaction using nucleocapsid-specific primers and spike-specific primers. Prevalence in broiler flocks was 39.4% (26/66) and in local chicken flocks was 11.9% (5/42). Several IBVs similar to Japan were detected in Taiwan. One-direction neutralization revealed that the reference antisera did not offer protection against the IBVs similar to those from Japan.

Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture.

PubMed

Jiang, Hong; Du, Hong; Wang, Li M; Wang, Ping Z; Bai, Xue F

2016-01-01

Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS), which is a zoonosis endemic in eastern Asia, especially in China. The reservoir host of HTNV is field mouse (Apodemus agraricus). The main manifestation of HFRS, including acute kidney injury, increases vascular permeability, and coagulation abnormalities. In this paper, we review the current knowledge of the pathogenesis of HFRS including virus factor, immunity factor and host genetic factors. Furthermore, the treatment and prevention will be discussed.

Hemorrhagic Fever with Renal Syndrome: Pathogenesis and Clinical Picture

PubMed Central

Jiang, Hong; Du, Hong; Wang, Li M.; Wang, Ping Z.; Bai, Xue F.

2016-01-01

Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS), which is a zoonosis endemic in eastern Asia, especially in China. The reservoir host of HTNV is field mouse (Apodemus agraricus). The main manifestation of HFRS, including acute kidney injury, increases vascular permeability, and coagulation abnormalities. In this paper, we review the current knowledge of the pathogenesis of HFRS including virus factor, immunity factor and host genetic factors. Furthermore, the treatment and prevention will be discussed. PMID:26870699

Measles virus genetic evolution throughout an imported epidemic outbreak in a highly vaccinated population.

PubMed

Muñoz-Alía, Miguel Ángel; Fernández-Muñoz, Rafael; Casasnovas, José María; Porras-Mansilla, Rebeca; Serrano-Pardo, Ángela; Pagán, Israel; Ordobás, María; Ramírez, Rosa; Celma, María Luisa

2015-01-22

Measles virus circulates endemically in African and Asian large urban populations, causing outbreaks worldwide in populations with up-to-95% immune protection. We studied the natural genetic variability of genotype B3.1 in a population with 95% vaccine coverage throughout an imported six month measles outbreak. From first pass viral isolates of 47 patients we performed direct sequencing of genomic cDNA. Whilst no variation from index case sequence occurred in the Nucleocapsid gene hyper-variable carboxy end, in the Hemagglutinin gene, main target for neutralizing antibodies, we observed gradual nucleotide divergence from index case along the outbreak (0% to 0.380%, average 0.138%) with the emergence of transient and persistent non-synonymous and synonymous mutations. Little or no variation was observed between the index and last outbreak cases in Phosphoprotein, Nucleocapsid, Matrix and Fusion genes. Most of the H non-synonymous mutations were mapped on the protein surface near antigenic and receptors binding sites. We estimated a MV-Hemagglutinin nucleotide substitution rate of 7.28 × 10-6 substitutions/site/day by a Bayesian phylogenetic analysis. The dN/dS analysis did not suggest significant immune or other selective pressures on the H gene during the outbreak. These results emphasize the usefulness of MV-H sequence analysis in measles epidemiological surveillance and elimination programs, and in detection of potentially emergence of measles virus neutralization-resistant mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice.

PubMed

Zheng, Nuoyan; Xia, Ran; Yang, Cuiping; Yin, Bojiao; Li, Yin; Duan, Chengguo; Liang, Liming; Guo, Huishan; Xie, Qi

2009-08-06

Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79 microg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-gamma and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus.

PubMed

Reichelt, Mike; Wang, Li; Sommer, Marvin; Perrino, John; Nour, Adel M; Sen, Nandini; Baiker, Armin; Zerboni, Leigh; Arvin, Ann M

2011-02-03

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant

Virus-like particles as nanovaccine candidates

NASA Astrophysics Data System (ADS)

Guillen, G.; Aguilar, J. C.; Dueñas, S.; Hermida, L.; Iglesias, E.; Penton, E.; Lobaina, Y.; Lopez, M.; Mussachio, A.; Falcon, V.; Alvarez, L.; Martinez, G.; Gil, L.; Valdes, I.; Izquierdo, A.; Lazo, L.; Marcos, E.; Guzman, G.; Muzio, V.; Herrera, L.

2013-03-01

The existing vaccines are mainly limited to the microorganisms we are able to culture and produce and/or to those whose killing is mediated by humoral response (antibody mediated). It has been more difficult to develop vaccines capable of inducing a functional cellular response needed to prevent or cure chronic diseases. New strategies should be taken into account in the improvement of cell-based immune responses in order to prevent and control the infections and eventually clear the virus. Preclinical and clinical results with vaccine candidates developed as a vaccine platform based on virus-like particles (VLPs) evidenced their ability to stimulate mucosal as well as systemic immunity. Particles based on envelope, membrane or nucleocapsid microbial proteins induce a strong immune response after nasal or parenteral administration in mice, non-human primates and humans. In addition, the immune response obtained was modulated in a Th1 sense. The VLPs were also able to immunoenhance the humoral and cellular immune responses against several viral pathogens. Studies in animals and humans with nasal and systemic formulations evidenced that it is possible to induce functional immune response against HBV, HCV, HIV and dengue virus. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology, 30 October - 2 November 2012, Ha Long, Vietnam.

Methods for the analysis of HIV-1 nucleocapsid protein interactions with oligonucleotides.

PubMed

Stephen, Andrew G; Fisher, Robert J

2009-01-01

HIV-1 Nucleocapsid protein (NC) is a small basic protein that contains two retroviral zinc fingers. It is a highly effective nucleic acid chaperone that plays a critical role in viral replication acting as a cofactor in reverse transcription as well as other aspects of the viral lifecycle. We have used a variety of biophysical techniques to characterize the high affinity binding of NC to a short deoxyoligonucleotide (d(TG)(4)). Here we outline in detail the use of fluorescence anisotropy and surface plasmon resonance spectroscopy to study the binding of NC to d(TG)(4).

New strains of rabies-related viruses isolated from bats in the Ukraine.

PubMed

Selimov, M A; Smekhov, A M; Antonova, L A; Shablovskaya, E A; King, A A; Kulikova, L G

1991-05-01

Two strains (UB-1 and UB-2) of rabies-related viruses were isolated from the brain of Nyctalus noctula and Vespertilio murinus captured from the hollows of tall trees on the left bank of Pripyat river in the Volynsky region of Ukrainian S.S.R. The viruses were isolated by means of intracerebral inoculation to white mice. The isolates were identified as rabies-related viruses of Duvenhage type in an indirect test of fluorescent antibodies with the panels of nucleocapsid monoclonal antibodies (NC Mab) provided by Wistar Institute (Philadelphia) and by Central Veterinary Laboratory (CVL, Weybridge). During the typing with the Wistar panel of NC Mab complete antigenic similarity was established between the newly isolated strain and Yuli virus. The reaction with CVL NC Mab revealed group-specific antigenic similarity between Yuli virus on one hand, Duvenhage-6 and Duvenhage-66 on the other hand, as well as between UB-1 and UB-2 and Duvenhage-26. The reaction with antibodies to clones DB-3,4,6,9, and 10 detected antigenic similarity between the viruses of chiropteric origin isolated in the U.S.S.R., North-West Europe as well in Africa, although some differences were discovered. Yuli, UB-1, and UB-2 viruses isolated in the U.S.S.R. were proved to belong to Duvenhage group of viruses (serotype 4).

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds

The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient.

PubMed

Cox, Robert M; Krumm, Stefanie A; Thakkar, Vidhi D; Sohn, Maximilian; Plemper, Richard K

2017-02-01

The paramyxovirus RNA-dependent RNA-polymerase (RdRp) complex loads onto the nucleocapsid protein (N)-encapsidated viral N:RNA genome for RNA synthesis. Binding of the RdRp of measles virus (MeV), a paramyxovirus archetype, is mediated through interaction with a molecular recognition element (MoRE) located near the end of the carboxyl-terminal Ntail domain. The structurally disordered central Ntail section is thought to add positional flexibility to MoRE, but the functional importance of this Ntail region for RNA polymerization is unclear. To address this question, we dissected functional elements of Ntail by relocating MoRE into the RNA-encapsidating Ncore domain. Linker-scanning mutagenesis identified a microdomain in Ncore that tolerates insertions. MoRE relocated to Ncore supported efficient interaction with N, MoRE-deficient Ntails had a dominant-negative effect on bioactivity that was alleviated by insertion of MoRE into Ncore, and recombinant MeV encoding N with relocated MoRE grew efficiently and remained capable of mRNA editing. MoRE in Ncore also restored viability of a recombinant lacking the disordered central Ntail section, but this recombinant was temperature-sensitive, with reduced RdRp loading efficiency and a flattened transcription gradient. These results demonstrate that virus replication requires high-affinity RdRp binding sites in N:RNA, but productive RdRp binding is independent of positional flexibility of MoRE and cis-acting elements in Ntail. Rather, the disordered central Ntail section independent of the presence of MoRE in Ntail steepens the paramyxovirus transcription gradient by promoting RdRp loading and preventing the formation of nonproductive polycistronic viral mRNAs. Disordered Ntails may have evolved as a regulatory element to adjust paramyxovirus gene expression.

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation.

PubMed

Zhao, Qiong; Hu, Zhanying; Cheng, Junjun; Wu, Shuo; Luo, Yue; Chang, Jinhong; Hu, Jianming; Guo, Ju-Tao

2018-07-01

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which

Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

NASA Astrophysics Data System (ADS)

Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

Biochemical and structural studies of the oligomerization domain of the Nipah virus phosphoprotein: evidence for an elongated coiled-coil homotrimer.

PubMed

Blocquel, David; Beltrandi, Matilde; Erales, Jenny; Barbier, Pascale; Longhi, Sonia

2013-11-01

Nipah virus (NiV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The NiV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the polymerase for transcription and replication. The polymerase is recruited onto the nucleocapsid via its cofactor, the phosphoprotein (P). The NiV P protein has a modular organization, with alternating disordered and ordered domains. Among these latter, is the P multimerization domain (PMD) that was predicted to adopt a coiled-coil conformation. Using both biochemical and biophysical approaches, we show that NiV PMD forms a highly stable and elongated coiled-coil trimer, a finding in striking contrast with respect to the PMDs of Paramyxoviridae members investigated so far that were all found to tetramerize. The present results therefore represent the first report of a paramyxoviral P protein forming trimers. © 2013 Elsevier Inc. All rights reserved.

Measles inclusion-body encephalitis caused by the vaccine strain of measles virus.

PubMed

Bitnun, A; Shannon, P; Durward, A; Rota, P A; Bellini, W J; Graham, C; Wang, E; Ford-Jones, E L; Cox, P; Becker, L; Fearon, M; Petric, M; Tellier, R

1999-10-01

We report a case of measles inclusion-body encephalitis (MIBE) occurring in an apparently healthy 21-month-old boy 8.5 months after measles-mumps-rubella vaccination. He had no prior evidence of immune deficiency and no history of measles exposure or clinical disease. During hospitalization, a primary immunodeficiency characterized by a profoundly depressed CD8 cell count and dysgammaglobulinemia was demonstrated. A brain biopsy revealed histopathologic features consistent with MIBE, and measles antigens were detected by immunohistochemical staining. Electron microscopy revealed inclusions characteristic of paramyxovirus nucleocapsids within neurons, oligodendroglia, and astrocytes. The presence of measles virus in the brain tissue was confirmed by reverse transcription polymerase chain reaction. The nucleotide sequence in the nucleoprotein and fusion gene regions was identical to that of the Moraten and Schwarz vaccine strains; the fusion gene differed from known genotype A wild-type viruses.

A Snapshot Avian Surveillance Reveals West Nile Virus and Evidence of Wild Birds Participating in Toscana Virus Circulation.

PubMed

Hacioglu, Sabri; Dincer, Ender; Isler, Cafer Tayer; Karapinar, Zeynep; Ataseven, Veysel Soydal; Ozkul, Aykut; Ergunay, Koray

2017-10-01

Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts for viruses, dissemination vehicles for the vectors, and sources of emerging strains in cross-species transmission. Turkey provides diverse habitats for a variety of wild birds and is located along major bird migration routes. This study was undertaken to provide a cross-sectional screening of avian specimens for a spectrum of vector-borne viruses. The specimens were collected in Hatay province, in the Mediterranean coast of the Anatolian peninsula, located in the convergence zone of the known migration routes. Generic PCR assays were used for the detection of members of Nairovirus, Flavivirus, and Phlebovirus genera of Flaviviridae and Bunyaviridae families. The circulating viruses were characterized via sequencing and selected specimens were inoculated onto Vero cell lines for virus isolation. Specimens from 72 wild birds belonging in 8 orders and 14 species were collected. A total of 158 specimens that comprise 32 sera (20.3%) from 7 species and 126 tissues (79.7%) from 14 species were screened. Eight specimens (8/158, 5%), obtained from 4 individuals (4/72, 5.5%), were positive. West Nile virus (WNV) lineage 1 sequences were characterized in the spleen, heart, and kidney tissues from a lesser spotted eagle (Clanga pomarina), which distinctly clustered from sequences previously identified in Turkey. Toscana virus (TOSV) genotype A and B sequences were identified in brain and kidney tissues from a greater flamingo (Phoenicopterus roseus), a great white pelican (Pelecanus onocrotalus), and a black stork (Ciconia nigra), without successful virus isolation. Partial amino acid sequences of the viral nucleocapsid protein revealed previously unreported substitutions. This study documents the involvement of avians in WNV dispersion in Anatolia as well in TOSV life cycle.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.

PubMed

Wang, Qiang; Ma, Xiaonan; Qian, ShaSha; Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O; Li, Zhenghe

2015-10-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

PubMed Central

Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O.; Li, Zhenghe

2015-01-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses. PMID:26484673

The nucleocapsid protein of measles virus blocks host interferon response

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

Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira

2012-03-01

Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-{alpha}/{beta} and {gamma}-antagonist as strong as Pmore » gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.« less

Pathogenic effects of Rift Valley fever virus NSs gene are alleviated in cultured cells by expressed antiviral short hairpin RNAs.

PubMed

Scott, Tristan; Paweska, Janusz T; Arbuthnot, Patrick; Weinberg, Marc S

2012-01-01

Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, may cause severe hepatitis, encephalitis and haemorrhagic fever in humans. There are currently no available licensed vaccines or therapies to treat the viral infection in humans. RNA interference (RNAi)-based viral gene silencing offers a promising approach to inhibiting replication of this highly pathogenic virus. The small (S) segment of the RVFV tripartite genome carries the genetic determinates for pathogenicity during infection. This segment encodes the non-structural S (NSs) and essential nucleocapsid (N) genes. To advance RNAi-based inhibition of RVFV replication, we designed several Pol III short hairpin RNA (shRNA) expression cassettes against the NSs and N genes, including a multimerized plasmid vector that included four shRNA expression cassettes. Effective target silencing was demonstrated using full- and partial-length target reporter assays, and confirmed by western blot analysis of exogenous N and NSs expression. Small RNA northern blots showed detectable RNAi guide strand formation from single and multimerized shRNA constructs. Using a cell culture model of RVFV replication, shRNAs targeting the N gene decreased intracellular nucleocapsid protein concentration and viral replication. The shRNAs directed against the NSs gene reduced NSs protein concentrations and alleviated NSs-mediated cytotoxicity, which may be caused by host transcription suppression. These data are the first demonstration that RNAi activators have a potential therapeutic benefit for countering RVFV infection.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

PubMed Central

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei

2015-01-01

ABSTRACT Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

PubMed

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei; Wang, Hualin; Lou, Zhiyong

2016-01-15

Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

PubMed

Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

2001-04-06

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

Development and Application of Quantitative Detection Method for Viral Hemorrhagic Septicemia Virus (VHSV) Genogroup IVa

PubMed Central

Kim, Jong-Oh; Kim, Wi-Sik; Kim, Si-Woo; Han, Hyun-Ja; Kim, Jin Woo; Park, Myoung Ae; Oh, Myung-Joo

2014-01-01

Viral hemorrhagic septicemia virus (VHSV) is a problematic pathogen in olive flounder (Paralichthys olivaceus) aquaculture farms in Korea. Thus, it is necessary to develop a rapid and accurate diagnostic method to detect this virus. We developed a quantitative RT-PCR (qRT-PCR) method based on the nucleocapsid (N) gene sequence of Korean VHSV isolate (Genogroup IVa). The slope and R2 values of the primer set developed in this study were −0.2928 (96% efficiency) and 0.9979, respectively. Its comparison with viral infectivity calculated by traditional quantifying method (TCID50) showed a similar pattern of kinetic changes in vitro and in vivo. The qRT-PCR method reduced detection time compared to that of TCID50, making it a very useful tool for VHSV diagnosis. PMID:24859343

Shimoni bat virus, a new representative of the Lyssavirus genus.

PubMed

Kuzmin, Ivan V; Mayer, Anne E; Niezgoda, Michael; Markotter, Wanda; Agwanda, Bernard; Breiman, Robert F; Rupprecht, Charles E

2010-05-01

During 2009, 616 bats representing at least 22 species were collected from 10 locations throughout Kenya. A new lyssavirus, named Shimoni bat virus (SHIBV), was isolated from the brain of a dead Commerson's leaf-nosed bat (Hipposideros commersoni), found in a cave in the coastal region of Kenya. Genetic distances and phylogenetic reconstructions, implemented for each gene and for the concatenated alignment of all five structural genes (N, P, M, G and L), demonstrated that SHIBV cannot be identified with any of the existing species, but rather should be considered an independent species within phylogroup II of the Lyssavirus genus, most similar to Lagos bat virus (LBV). Antigenic reaction patterns with anti-nucleocapsid monoclonal antibodies corroborated these distinctions. In addition, new data on the diversity of LBV suggests that this species may be subdivided quantitatively into three separate genotypes. However, the identity values alone are not considered sufficient criteria for demarcation of new species within LBV. (c) 2010 Elsevier B.V. All rights reserved.

Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia

USGS Publications Warehouse

Rudakova, S.L.; Kurath, G.; Bochkova, E.V.

2007-01-01

Infectious hematopoietic necrosis virus (IHNV) is a well known rhabdoviral pathogen of salmonid fish in North America that has become established in Asia and Europe. On the Pacific coast of Russia, IHNV was first detected in hatchery sockeye from the Kamchatka Peninsula in 2001. Results of virological examinations of over 10 000 wild and cultured salmonid fish from Kamchatka during 1996 to 2005 revealed IHNV in several sockeye salmon Oncorhynchus nerka populations. The virus was isolated from spawning adults and from juveniles undergoing epidemics in both hatchery and wild sockeye populations from the Bolshaya watershed. No virus was detected in 2 other water-sheds, or in species other than sockeye salmon. Genetic typing of 8 virus isolates by seguence analysis of partial glycoprotein and nucleocapsid genes revealed that they were genetically homogeneous and fell within the U genogroup of IHNV. In phylogenetic analyses, the Russian IHNV sequences were indistinguishable from the sequences of North American U genogroup isolates that occur throughout Alaska, British Columbia, Washington, and Oregon. The high similarity, and in some cases identity, between Russian and North American IHNV isolates suggests virus transmission or exposure to a common viral reservoir in the North Pacific Ocean. ?? Inter-Research 2007.

Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA.

PubMed

Hagiwara, Kyoji; Sato, Hiroki; Inoue, Yoshihisa; Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Fujita, Kentaro; Fukuda, Hiroyuki; Takamura, Chizuko; Kozuka-Hata, Hiroko; Oyama, Masaaki; Sugano, Sumio; Ohmi, Shinobu; Kai, Chieko

2008-05-01

We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N-RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C-terminal region from the core region, phosphorylated amino acids were assayed using MALDI-TOF/TOF and ESI-Q-TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with (32)P demonstrated that these two sites are the major phosphorylated sites within the MV-N protein. In transcriptional analysis using negative-strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild-type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.

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.

Vaccination of calves using the BRSV nucleocapsid protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects the lungs against BRSV replication and pathology.

PubMed

Letellier, Carine; Boxus, Mathieu; Rosar, Laurent; Toussaint, Jean-François; Walravens, Karl; Roels, Stefan; Meyer, Gilles; Letesson, Jean-Jacques; Kerkhofs, Pierre

2008-09-02

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and exacerbation of subsequent RSV disease have occurred. In order to circumvent these problems, calves were vaccinated with the nucleocapsid protein, known to be a major target of CD8(+) T cells in cattle. This was performed according to a DNA prime-protein boost strategy. The results showed that DNA vaccination primed a specific T-cell-mediated response, as indicated by both a lymphoproliferative response and IFN-gamma production. These responses were enhanced after protein boost. After challenge, mock-vaccinated calves displayed gross pneumonic lesions and viral replication in the lungs. In contrast, calves vaccinated by successive administrations of plasmid DNA and protein exhibited protection against the development of pneumonic lesions and the viral replication in the BAL fluids and the lungs. The protection correlated to the cell-mediated immunity and not to the antibody response.

The integrity of PRRSV nucleocapsid protein is necessary for up-regulation of optimal interleukin-10 through NF-κB and p38 MAPK pathways in porcine alveolar macrophages.

PubMed

Yu, Jiang; Liu, Yanyan; Zhang, Yuyu; Zhu, Xiwang; Ren, Sufang; Guo, Lihui; Liu, Xing; Sun, Wenbo; Chen, Zhi; Cong, Xiaoyan; Chen, Lei; Shi, Jianli; Du, Yijun; Li, Jun; Wu, Jiaqiang; Wang, Jinbao

2017-08-01

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease, has been constantly causing huge economic losses all over the world. PRRS virus (PRRSV) infection results in immunosuppression and IL-10 up-regulation. The relationship between them is still in dispute. Previous studies demonstrated the protein of PRRSV nucleocapsid (N) protein is able to up-regulate IL-10, yet the underlying molecular mechanisms remain unknown. In this study, the expression kinetics of IL-10 up-regulation induced by PRRSV N protein were analyzed in immortalized porcine alveolar macrophages (PAMs). N protein induced IL-10 expression in a time- and dose-dependent manner. Inhibition experiments of signaling pathways suggested NF-κB and p38 MAPK pathways are both involved in N protein-induced IL-10 up-regulation. Besides, the integrity of N protein is essential for significant IL-10 up-regulation. This research is beneficial for further understanding of the interplay between PRRSV and host immune system. Copyright © 2017. Published by Elsevier Ltd.

Breach of the nuclear lamina during assembly of herpes simplex viruses.

PubMed

Morrison, Lynda A; DeLassus, Gregory S

2011-01-01

Beneath the inner nuclear membrane lies the dense meshwork of the nuclear lamina, which provides structural support for the nuclear envelope and serves as an important organizing center for a number of nuclear and cytoplasmic constituents and processes. Herpesviruses have a significant and wide-ranging impact on human health, and their capacity to replicate and cause disease includes events that occur in the host cell nucleus. Herpesviruses begin assembly of progeny virus in the nuclei of infected cells and their capsids must escape the confines of the nucleus by budding through the inner nuclear membrane (INM) to proceed with later stages of virion assembly and egress. Access of viral capsids to the INM thus necessitates disruption of the dense nuclear lamina layer. We review herpesvirus effects on the nuclear lamina and in particular the roles of the herpes simplex virus-encoded nuclear envelope complex and viral kinases on lamin phosphorylation, dissociation, and nucleocapsid envelopment at the INM.

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.

PubMed

Bharat, Tanmay A M; Noda, Takeshi; Riches, James D; Kraehling, Verena; Kolesnikova, Larissa; Becker, Stephan; Kawaoka, Yoshihiro; Briggs, John A G

2012-03-13

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus.

PubMed

Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

2016-05-01

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus

The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

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

Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.

Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures ofmore » the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.« less

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

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

PubMed

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

Strains of infectious hematopoietic necrosis (IHN) virus may be identified by structural protein differences

USGS Publications Warehouse

Leong, J.C.; Hsu, Ya Li; Engelking, H. Mark; Mulcahy, Daniel M.; Anderson, D.P.

1981-01-01

The development of an effective vaccine to infectious hematopoietic necrosis virus (IHNV) in fish requires a knowledge of the virus serotypes in nature. At least two serotypes were found among three IHNV strains (12). Attempts in our laboratory to extend this study with additional virus strains by classical immunological techniques were unsatisfactory. Thus, we sought another method for comparing IHNV strains. We report here that different strains of IHNV can be distinguished by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the viron polypeptides. Major strain differences were noted in the molecular weights of the envelope glycoprotein, G, and the nucleocapsid protein, N.The strains of IHNV were established from samples taken at several locations in California, Oregon, Washington, and Alaska. These strains were also characterized by growth rate at 15° and 18°C and average plaque size. Distinct differences in growth rate at the two temperatures were found for the California strains. The California strains were distinguished from the other strains by the production of minute plaques.

Sequence diversity of wheat mosaic virus isolates.

PubMed

Stewart, Lucy R

2016-02-02

Wheat mosaic virus (WMoV), transmitted by eriophyid wheat curl mites (Aceria tosichella) is the causal agent of High Plains disease in wheat and maize. WMoV and other members of the genus Emaravirus evaded thorough molecular characterization for many years due to the experimental challenges of mite transmission and manipulating multisegmented negative sense RNA genomes. Recently, the complete genome sequence of a Nebraska isolate of WMoV revealed eight segments, plus a variant sequence of the nucleocapsid protein-encoding segment. Here, near-complete and partial consensus sequences of five more WMoV isolates are reported and compared to the Nebraska isolate: an Ohio maize isolate (GG1), a Kansas barley isolate (KS7), and three Ohio wheat isolates (H1, K1, W1). Results show two distinct groups of WMoV isolates: Ohio wheat isolate RNA segments had 84% or lower nucleotide sequence identity to the NE isolate, whereas GG1 and KS7 had 98% or higher nucleotide sequence identity to the NE isolate. Knowledge of the sequence variability of WMoV isolates is a step toward understanding virus biology, and potentially explaining observed biological variation. Published by Elsevier B.V.

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

PubMed Central

Överby, A. K.; Pettersson, R. F.; Grünewald, K.; Huiskonen, J. T.

2008-01-01

Bunyaviridae is a large family of viruses that have gained attention as “emerging viruses” because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or “spikes,” were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry. PMID:18272496

Characterization of the size distribution and aggregation of virus-like nanoparticles used as active ingredients of the HeberNasvac therapeutic vaccine against chronic hepatitis B

NASA Astrophysics Data System (ADS)

Lopez, Matilde; Rodriguez, Elias Nelson; Lobaina, Yadira; Musacchio, Alexis; Falcon, Viviana; Guillen, Gerardo; Aguilar, Julio C.

2017-06-01

The use of virus-like particles (VLPs) as antigens constitutes a well established strategy in preventive vaccination. These non-infective particles have a composition, size, and structure favoring their interaction and processing by the immune system. Recombinant viral nucleocapsids encapsulating bacterial nucleic acids result in potent Th1-driving immunogens. Several antigens have been coadministered with VLPs or conjugated to them to further increase their immunogenicity. In the present work we characterize the size distribution of two different recombinant VLPs obtained as components of HeberNasvac, a novel therapeutic vaccine recently registered to treat chronic hepatitis B. The vaccine ingredients, hepatitis B virus surface and nucleocapsid antigens (HBsAg and HBcAg, respectively) and the vaccine formulation, were evaluated using dynamic light scattering (DLS), transmission electron microscopy (TEM) and light obscuration technology. The results demonstrate that both antigens are nanoparticles with sizes ranging between 20-30 nm, in line with reports in the literature. In addition, DLS studies evidenced the capacity of both antigens to form homologous and heterologous aggregates, both as active ingredients as well as being part of the final product. The evaluation of subvisible particles in HeberNasvac formulation fulfills the requirements in terms of quantity and size established for parenteral pharmaceutical compositions. Invited talk at 8th Int. Workshop on Advanced Materials Science and Nanotechnology (IWAMSN2016) (Ha Long City, Vietnam, 8-12 November 2016)

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis

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

Baczko, K.; Liebert, U.G.; Billeter, M.

1986-08-01

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the bran sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in themore » different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.« less

[Epidemiology of hantaviruses in Baden-Wurttemberg].

PubMed

Kimmig, P; Silva-González, R; Backe, H; Brockmann, S; Oehme, R; Ernst, E; Mackenstedt, U

2001-02-01

Hantavirus, originally named after the Hantaan River in Korea, is the aetiologic agent for the Hemorrhagic Fever with Renal Syndrome (HFRS) in the asian region, in the Americas for the Hantavirus Pulmonary Syndrome (HPS). In Middle Europe hantaviruses are responsible for the "Nephropathia Epidemica" (NE), a mild form of HFRS. Hantaviruses belong to the family of Bunyaviridae. Like other members of this family their genome consists of three segments of single stranded RNA (ss-RNA) leading to various subtypes, strongly associated with different rodent hosts. There are two major groups, the hantaan lineage harbored by murine rodents and the Puumala lineage harbored by arvicolidae ("old world") and sigmodontidae ("new world"). Infected rodents may develop chronic infections for months or even life-long and may shed infectious virus with urine and feces. The primary mode of infection of man occurs by inhaling contaminated aerosols or soil particles. The collection of epidemiologic data in the state of Baden-Württemberg was realized in three different steps: Collection and localisation of clinical cases (n = 62): A concentration of clinical cases in the middle of the state was found. The examination of the seroprevalence of exposed persons: By the examination of 4000 sera from forest workers, a seroprevalence with an average of 2.1% was found. In the districts of Reutlingen and Tübingen seroprevalences up to 9% were found. This leads to the assumption that there are endemic areas. Epidemiologic studies of reservoir hosts: Serologic surveys of rodents (n = 1150) in the described areas yielded to a seroprevalence up to 10-30%. Virus carriers were determined with RT-PCR and nested-PCR testing. The prevalence in the rodent population showed an average of 10%. The isolated subtypes were all identified as members of the Puumala-lineage. The origin of sporadic infections with Hantavirus of the Hantaan-lineage in Baden-Wuerttemberg is still unknown.

Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.

PubMed Central

Brulé, Fabienne; Marquet, Roland; Rong, Liwei; Wainberg, Mark A; Roques, Bernard P; Le Grice, Stuart F J; Ehresmann, Bernard; Ehresmann, Chantal

2002-01-01

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo. PMID:11873759

Development of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus.

PubMed

Rao, Xueqin; Sun, Jie

2015-09-01

Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus , causes significant loss in Cucurbitaceae plants. Development of a highly sensitive and reliable detection method for WSMoV. Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27 watermelon samples showing WSMoV infection symptoms. The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and melting curves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Capsicum chlorosis virus (genus Tospovirus ) and Cucumber mosaic virus (genus Cucumovirus). Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%. A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detection of WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.

Electron Tomography Reveals the Steps in Filovirus Budding

PubMed Central

Welsch, Sonja; Kolesnikova, Larissa; Krähling, Verena; Riches, James D.; Becker, Stephan; Briggs, John A. G.

2010-01-01

The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses. PMID:20442788

Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

PubMed

Lasecka, Lidia; Bin-Tarif, Abdelghani; Bridgen, Anne; Juleff, Nicholas; Waters, Ryan A; Baron, Michael D

2015-01-01

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.

Structure of the recombinant alphavirus Western equine encephalitis virus revealed by cryoelectron microscopy.

PubMed

Sherman, Michael B; Weaver, Scott C

2010-10-01

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV's structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.

Measles Virus Nucleocapsid (MVNP) Gene Expression and RANK Receptor Signaling in Osteoclast Precursors,Osteoclast Inhibitors Peptide Therapy for Pagets Disease

DTIC Science & Technology

2004-10-01

Anderson DC, Sharpe PT. 1992. Canine distemper virus transcripts sequenced from pagetic bone. Bone Miner 19:159–174. Gori F, Hofbauer LC, Dunstan CR...clast formation in Paget’s disease. QJM 95:233–240. Ooi CG, Walsh CA, Gallagher JA, Fraser WD. 2000. Absence of measles virus and canine distemper virus...techniques. Furthermore, it has also been de- monstrated that infecting canine bone marrow cells with CDV results in development of multi- nucleated cells

A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus.

PubMed

Pappi, Polyxeni G; Dovas, Chrysostomos I; Efthimiou, Konstantinos E; Maliogka, Varvara I; Katis, Nikolaos I

2013-08-01

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.

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.

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

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

PubMed Central

Boregowda, Rajeev; Sohn, Ji A.; Ledesma, Felipe Cortes; Caldecott, Keith W.; Seeger, Christoph; Hu, Jianming

2015-01-01

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5’ end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5’ DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5’ DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5’ end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo. PMID:26079492

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

The complete genome structure and phylogenetic relationship of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Morzunov , Sergey P.; Winton, James R.; Nichol, Stuart T.

1995-01-01

Infectious hematopoietic necrosis virus (IHNV), a member of the family Rhabdoviridae, causes a severe disease with high mortality in salmonid fish. The nucleotide sequence (11, 131 bases) of the entire genome was determined for the pathogenic WRAC strain of IHNV from southern Idaho. This allowed detailed analysis of all 6 genes, the deduced amino acid sequences of their encoded proteins, and important control motifs including leader, trailer and gene junction regions. Sequence analysis revealed that the 6 virus genes are located along the genome in the 3′ to 5′ order: nucleocapsid (N), polymerase-associated phosphoprotein (P or M1), matrix protein (M or M2), surface glycoprotein (G), a unique non-virion protein (NV) and virus polymerase (L). The IHNV genome RNA was found to have highly complementary termini (15 of 16 nucleotides). The gene junction regions display the highly conserved sequence UCURUC(U)7RCCGUG(N)4CACR (in the vRNA sense), which includes the typical rhabdovirus transcription termination/polyadenylation signal and a novel putative transcription initiation signal. Phylogenetic analysis of M, G and L protein sequences allowed insights into the evolutionary and taxonomic relationship of rhabdoviruses of fish relative to those of insects or mammals, and a broader sense of the relationship of non-segmented negative-strand RNA viruses. Based on these data, a new genus, piscivirus, is proposed which will initially contain IHNV, viral hemorrhagic septicemia virus and Hirame rhabdovirus.

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

PubMed Central

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc

2017-01-01

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China

PubMed Central

2011-01-01

A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China. PMID:22087872

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China.

PubMed

Tan, Bin; Wen, Yong-Jun; Wang, Feng-Xue; Zhang, Shu-Qin; Wang, Xiu-Dong; Hu, Jia-Xin; Shi, Xin-Chuan; Yang, Bo-Chao; Chen, Li-Zhi; Cheng, Shi-Peng; Wu, Hua

2011-11-16

A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.

Manual of Hemorrhagic Fever with Renal Syndrome

DTIC Science & Technology

1989-04-01

Reference and Research * ~(Hemorrhagic fever with renal syndrome) Institute for Vral Diseases, Korea University Preface The discovery of Hantaan virus...in 1976 launched a new era in the study of hemorrhagic fever with renal syndrome (HFRS) throughout the world. Accumulated data indicate that there...HFRS that has made all of this possible. April 1989 H. W. Lee M.D. J. M. Dalrymple Ph.D. Foreword Haemorrhagic Fever with Renal Syndrome (HFRS) is

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

USGS Publications Warehouse

Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

2013-01-01

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

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

South, T.L.; Blake, P.R.; Hare, D.R.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhausermore » effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.« less

Rapid Nipah virus entry into the central nervous system of hamsters via the olfactory route

PubMed Central

Munster, Vincent J.; Prescott, Joseph B.; Bushmaker, Trenton; Long, Dan; Rosenke, Rebecca; Thomas, Tina; Scott, Dana; Fischer, Elizabeth R.; Feldmann, Heinz; de Wit, Emmie

2012-01-01

Encephalitis is a hallmark of Nipah virus (NiV) infection in humans. The exact route of entry of NiV into the central nervous system (CNS) is unknown. Here, we performed a spatio-temporal analysis of NiV entry into the CNS of hamsters. NiV initially predominantly targeted the olfactory epithelium in the nasal turbinates. From there, NiV infected neurons were visible extending through the cribriform plate into the olfactory bulb, providing direct evidence of rapid CNS entry. Subsequently, NiV disseminated to the olfactory tubercle and throughout the ventral cortex. Transmission electron microscopy on brain tissue showed extravasation of plasma cells, neuronal degeneration and nucleocapsid inclusions in affected tissue and axons, providing further evidence for axonal transport of NiV. NiV entry into the CNS coincided with the occurrence of respiratory disease, suggesting that the initial entry of NiV into the CNS occurs simultaneously with, rather than as a result of, systemic virus replication. PMID:23071900

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

PubMed Central

Lasecka, Lidia; Bin-Tarif, Abdelghani; Bridgen, Anne; Juleff, Nicholas; Waters, Ryan A.; Baron, Michael D.

2015-01-01

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages. PMID:25905707

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein.

PubMed

Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R

2012-04-02

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

Creation of a Recombinant Rift Valley Fever Virus with a Two-Segmented Genome ▿ †

PubMed Central

Brennan, Benjamin; Welch, Stephen R.; McLees, Angela; Elliott, Richard M.

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed. PMID:21795328

HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

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

Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretchmore » of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.« less

HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

PubMed

Guo, Fang; Zhao, Qiong; Sheraz, Muhammad; Cheng, Junjun; Qi, Yonghe; Su, Qing; Cuconati, Andrea; Wei, Lai; Du, Yanming; Li, Wenhui; Chang, Jinhong; Guo, Ju-Tao

2017-09-01

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

Molecular detection and genetic analysis of Akabane virus genogroup Ib in small ruminants in Turkey.

PubMed

Şevik, Murat

2017-09-01

Akabane disease is a viral disease transmitted by biting midges and can cause teratogenic malformations in cattle, sheep and goats. Abortion outbreaks associated with arthrogryposis and cerebellar hypoplasia in two epidemiologically independent flocks were reported in the Mediterranean region of Turkey in 2015. Phylogenetic analysis based on nucleocapsid (N) gene sequences showed that the field isolates presented here belong to the genogroup Ib. The akabane virus (AKAV) field isolates analysed in this study displayed 6 new amino acid substitutions in N and non-structural protein chains compared with those of AKAV strains belonging to genogroup Ib. To the best of our knowledge, this is the first report on the presence of the AKAV genogroup Ib in Turkey.

Development of apple latent spherical virus-based vaccines against three tospoviruses.

PubMed

Taki, Ayano; Yamagishi, Noriko; Yoshikawa, Nobuyuki

2013-09-01

Apple latent spherical virus (ALSV) is characterized by its relatively broad host range, latency in most host plants, and ability to induce gene silencing in host plants. Herein, we focus on the above characteristic of ALSV and describe our development of ALSV vector vaccines against three tospoviruses, namely, Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Tomato spotted wilt virus (TSWV). DNA fragments of 201 nt of three tospovirus S-RNAs (silencing suppressor (NSS) and nucleocapsid protein (N) coding regions for each tospovirus) were inserted into an ALSV-RNA2 vector to obtain six types of ALSV vector vaccines. Nicotiana benthamiana plants at the five-leaf stage were inoculated with each ALSV vector vaccine and challenged with the corresponding tospovirus species. Tospovirus-induced symptoms and tospovirus replication after challenge were significantly suppressed in plants preinoculated with all ALSV vector vaccines having the N region fragment, indicating that strong resistance was acquired after infection with ALSV vector vaccines. On the other hand, cross protection was not significant in plants preinoculated with ALSV vectors having the NSs region fragment. Similarly, inoculation with an ALSV-RNA1 vector having the N region fragment in the 3'-noncoding region, but not the NSs region fragment, induced cross protection, indicating that cross protection is via RNA silencing, not via the function of the protein derived from the N region fragment. Our approach, wherein ALSV vectors and selected target inserts are used, enables rapid establishment of ALSV vector vaccines against many pathogenic RNA viruses with known sequences. Copyright © 2013 Elsevier B.V. All rights reserved.

Functional Characterization of the Putative Hepatitis B Virus Core Protein Late Domain Using Retrovirus Chimeras

PubMed Central

Garcia, Mayra L.; Reynolds, Tracy D.; Mothes, Walther; Robek, Michael D.

2013-01-01

The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP138) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells. PMID:24009707

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

PubMed

Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

2016-02-19

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

The Nucleotide Sequence and Spliced pol mRNA Levels of the Nonprimate Spumavirus Bovine Foamy Virus

PubMed Central

Holzschu, Donald L.; Delaney, Mari A.; Renshaw, Randall W.; Casey, James W.

1998-01-01

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein. PMID:9499074

Structure and assembly of a paramyxovirus matrix protein

PubMed Central

Battisti, Anthony J.; Meng, Geng; Winkler, Dennis C.; McGinnes, Lori W.; Plevka, Pavel; Steven, Alasdair C.; Morrison, Trudy G.; Rossmann, Michael G.

2012-01-01

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host’s cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly. PMID:22891297

Structure and assembly of a paramyxovirus matrix protein.

PubMed

Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

2012-08-28

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

[Transient expression and characterization of intracellular single chain Fv against the nucleocapsid protein of Hantavirus].

PubMed

Bai, Wen-tao; Xu, Zhi-kai; Zhang, Fang-lin; Luo, Wen; Liu, Yong; Wu, Xing-an; Yan, Yan

2004-11-01

To transiently express an intracellular single chain Fv of monoclonal antibody 1A8 against nucleocapsid protein of Hantavirus and characterize the immunological activities of the expressed products. COS-7 cells were transfected with mammalian expression vector 1A8-scFv-Ckappa/pCI-neo via lipofectin. The expressed product was identified by indirect immunofluorescence and immunoprecipitation. A diffuse pattern fluorescence was observed in less than 1% cytoplasm of transfected COS-7 cells. The binding of intracellular antibody fragments to NP antigen was confirmed by immunoprecipitation analysis. Transiently expressed single chain intrabodies can effectively target NP antigen in the cytoplasm. The present study may provide a new approach for treatment of Hantavirus.

Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR.

PubMed

Shin, Y; Mori, T; Okita, M; Gemma, T; Kai, C; Mikami, T

1995-06-01

For a rapid diagnosis of canine distemper virus (CDV) infection, the reverse transcription-PCR (RT-PCR) was carried out to detect CDV nucleoprotein (NP) gene from canine peripheral blood mononuclear cells (PBMCs). Two sets of primers were targeted to two regions of NP gene of CDV Onderstepoort strain. The NP gene fragments were well amplified by the RT-PCR in each of the RNA extracts from Vero cells infected with 6 laboratory strains of CDV including Onderstepoort strain, and from PBMCs of a dog experimentally infected with CDV. The amplified NP gene was detected in 17 of 32 samples from dogs which were clinically suspected for CDV infection at veterinary hospitals. No RT-PCR product was found in 52 samples from healthy dogs including 40 specific pathogen free beagles vaccinated with an attenuated live virus-vaccine for CDV and 12 stray dogs. The RT-PCR provides a fast, sensitive, and supplementary method for the diagnosis of CDV infection in dogs.

Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

A phylogenetic approach to following West Nile virus in Connecticut

PubMed Central

Anderson, John F.; Vossbrinck, Charles R.; Andreadis, Theodore G.; Iton, Anthony; Beckwith, William H.; Mayo, Donald R.

2001-01-01

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America. PMID:11606791

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

PubMed

Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

2017-01-01

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L

Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase.

PubMed

Brennan, Benjamin; Li, Ping; Elliott, Richard M

2011-12-01

The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding.

PubMed

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc; Zhong, Jin

2017-03-15

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced

Genomic analysis reveals Nairobi sheep disease virus to be highly diverse and present in both Africa, and in India in the form of the Ganjam virus variant.

PubMed

Yadav, Pragya D; Vincent, Martin J; Khristova, Marina; Kale, Charuta; Nichol, Stuart T; Mishra, Akhilesh C; Mourya, Devendra T

2011-07-01

Nairobi sheep disease (NSD) virus, the prototype tick-borne virus of the genus Nairovirus, family Bunyaviridae is associated with acute hemorrhagic gastroenteritis in sheep and goats in East and Central Africa. The closely related Ganjam virus found in India is associated with febrile illness in humans and disease in livestock. The complete S, M and L segment sequences of Ganjam and NSD virus and partial sequence analysis of Ganjam viral RNA genome S, M and L segments encoding regions (396 bp, 701 bp and 425 bp) of the viral nucleocapsid (N), glycoprotein precursor (GPC) and L polymerase (L) proteins, respectively, was carried out for multiple Ganjam virus isolates obtained from 1954 to 2002 and from various regions of India. M segments of NSD and Ganjam virus encode a large ORF for the glycoprotein precursor (GPC), (1627 and 1624 amino acids in length, respectively) and their L segments encode a very large L polymerase (3991 amino acids). The complete S, M and L segments of NSD and Ganjam viruses were more closely related to one another than to other characterized nairoviruses, and no evidence of reassortment was found. However, the NSD and Ganjam virus complete M segment differed by 22.90% and 14.70%, for nucleotide and amino acid respectively, and the complete L segment nucleotide and protein differing by 9.90% and 2.70%, respectively among themselves. Ganjam and NSD virus, complete S segment differed by 9.40-10.40% and 3.2-4.10 for nucleotide and proteins while among Ganjam viruses 0.0-6.20% and 0.0-1.4%, variation was found for nucleotide and amino acids. Ganjam virus isolates differed by up to 17% and 11% at the nucleotide level for the partial S and L gene fragments, respectively, with less variation observed at the deduced amino acid level (10.5 and 2%, S and L, respectively). However, the virus partial M gene fragment (which encodes the hypervariable mucin-like domain) of these viruses differed by as much as 56% at the nucleotide level. Phylogenetic

Epitope-based immunoinformatics and molecular docking studies of nucleocapsid protein and ovarian tumor domain of crimean-congo hemorrhagic Fever virus.

PubMed

Srinivasan, Pappu; Kumar, Sivakumar Prasanth; Karthikeyan, Muthusamy; Jeyakanthan, Jeyaram; Jasrai, Yogesh T; Pandya, Himanshu A; Rawal, Rakesh M; Patel, Saumya K

2011-01-01

Crimean-Congo hemorrhagic fever virus (CCHFV), the fatal human pathogen is transmitted to humans by tick bite, or exposure to infected blood or tissues of infected livestock. The CCHFV genome consists of three RNA segments namely, S, M, and L. The unusual large viral L protein has an ovarian tumor (OTU) protease domain located in the N terminus. It is likely that the protein may be autoproteolytically cleaved to generate the active virus L polymerase with additional functions. Identification of the epitope regions of the virus is important for the diagnosis, phylogeny studies, and drug discovery. Early diagnosis and treatment of CCHF infection is critical to the survival of patients and the control of the disease. In this study, we undertook different in silico approaches using molecular docking and immunoinformatics tools to predict epitopes which can be helpful for vaccine designing. Small molecule ligands against OTU domain and protein-protein interaction between a viral and a host protein have been studied using docking tools.

Hantavirus pulmonary syndrome.

PubMed

Macneil, Adam; Nichol, Stuart T; Spiropoulou, Christina F

2011-12-01

Hantavirus pulmonary syndrome (HPS) is a severe disease characterized by a rapid onset of pulmonary edema followed by respiratory failure and cardiogenic shock. The HPS associated viruses are members of the genus Hantavirus, family Bunyaviridae. Hantaviruses have a worldwide distribution and are broadly split into the New World hantaviruses, which includes those causing HPS, and the Old World hantaviruses [including the prototype Hantaan virus (HTNV)], which are associated with a different disease, hemorrhagic fever with renal syndrome (HFRS). Sin Nombre virus (SNV) and Andes virus (ANDV) are the most common causes of HPS in North and South America, respectively. Case fatality of HPS is approximately 40%. Pathogenic New World hantaviruses infect the lung microvascular endothelium without causing any virus induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of HPS. This article briefly reviews the knowledge on HPS-associated hantaviruses accumulated since their discovery, less than 20 years ago. Published by Elsevier B.V.

Nucleotide sequence of the coat protein gene of Lettuce big-vein virus.

PubMed

Sasaya, T; Ishikawa, K; Koganezawa, H

2001-06-01

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M(r) of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M(r) of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7.3 kb (ss-1) and 6.6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.

Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein.

PubMed

Mohamed, Maizan; McLees, Angela; Elliott, Richard M

2009-08-01

Viruses in the genus Orthobunyavirus, family Bunyaviridae, have a genome comprising three segments (called L, M, and S) of negative-sense RNA. Serological studies have classified the >170 named virus isolates into 18 serogroups, with a few additional as yet ungrouped viruses. Until now, molecular studies and full-length S-segment nucleotide sequences were available for representatives of eight serogroups; in all cases, the S segment encodes two proteins, N (nucleocapsid) and NSs (nonstructural), in overlapping open reading frames (ORFs) that are translated from the same mRNA. The NSs proteins of Bunyamwera virus (BUNV) and California serogroup viruses have been shown to play a role in inhibiting host cell mRNA and protein synthesis, thereby preventing induction of interferon (IFN). We have determined full-length sequences of the S segments of representative viruses in the Anopheles A, Anopheles B, and Tete serogroups, and we report here that these viruses do not show evidence of having an NSs ORF. In addition, these viruses have rather longer N proteins than those in the other serogroups. Most of the naturally occurring viruses that lack the NSs protein behaved like a recombinant BUNV with the NSs gene deleted in that they failed to prevent induction of IFN-beta mRNA. However, Tacaiuma virus (TCMV) in the Anopheles A serogroup inhibited IFN induction in a manner similar to that of wild-type BUNV, suggesting that TCMV has evolved an alternative mechanism, not involving a typical NSs protein, to antagonize the host innate immune response.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed

Darlix, J L; Gabus, C; Allain, B

1992-12-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed Central

Darlix, J L; Gabus, C; Allain, B

1992-01-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. Images PMID:1331519

Characterization of a Bombyx mori nucleopolyhedrovirus with Bmvp80 disruption.

PubMed

Tang, Xu-Dong; Xu, Yi-Peng; Yu, Lin-Lin; Lang, Guo-Jun; Tian, Cai-Hong; Zhao, Jin-Fang; Zhang, Chuan-Xi

2008-12-01

A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

PubMed

Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

2000-02-01

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

A novel Sin Nombre virus DNA vaccine and its inclusion in a candidate pan-hantavirus vaccine against hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS).

PubMed

Hooper, Jay W; Josleyn, Matthew; Ballantyne, John; Brocato, Rebecca

2013-09-13

Sin Nombre virus (SNV; family Bunyaviridae, genus Hantavirus) causes a hemorrhagic fever known as hantavirus pulmonary syndrome (HPS) in North America. There have been approximately 200 fatal cases of HPS in the United States since 1993, predominantly in healthy working-age males (case fatality rate 35%). There are no FDA-approved vaccines or drugs to prevent or treat HPS. Previously, we reported that hantavirus vaccines based on the full-length M gene segment of Andes virus (ANDV) for HPS in South America, and Hantaan virus (HTNV) and Puumala virus (PUUV) for hemorrhagic fever with renal syndrome (HFRS) in Eurasia, all elicited high-titer neutralizing antibodies in animal models. HFRS is more prevalent than HPS (>20,000 cases per year) but less pathogenic (case fatality rate 1-15%). Here, we report the construction and testing of a SNV full-length M gene-based DNA vaccine to prevent HPS. Rabbits vaccinated with the SNV DNA vaccine by muscle electroporation (mEP) developed high titers of neutralizing antibodies. Furthermore, hamsters vaccinated three times with the SNV DNA vaccine using a gene gun were completely protected against SNV infection. This is the first vaccine of any kind that specifically elicits high-titer neutralizing antibodies against SNV. To test the possibility of producing a pan-hantavirus vaccine, rabbits were vaccinated by mEP with an HPS mix (ANDV and SNV plasmids), or HFRS mix (HTNV and PUUV plasmids), or HPS/HFRS mix (all four plasmids). The HPS mix and HFRS mix elicited neutralizing antibodies predominantly against ANDV/SNV and HTNV/PUUV, respectively. Furthermore, the HPS/HFRS mix elicited neutralizing antibodies against all four viruses. These findings demonstrate a pan-hantavirus vaccine using a mixed-plasmid DNA vaccine approach is feasible and warrants further development. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phylogenetic Analysis of a Swine Influenza A(H3N2) Virus Isolated in Korea in 2012

PubMed Central

Park, Sehee; Lee, Sangmoo; Hwang, Min-Woong; Bae, Joon-Yong; Heo, Jun; Kim, Donghwan; Jang, Seok-Il; Kim, Kabsu; Park, Man-Seong

2014-01-01

Influenza A virus (IAV) can infect avian and mammalian species, including humans. The genome nature of IAVs may contribute to viral adaptation in different animal hosts, resulting in gene reassortment and the reproduction of variants with optimal fitness. As seen again in the 2009 swine-origin influenza A H1N1 pandemic, pigs are known to be susceptible to swine, avian, and human IAVs and can serve as a ‘mixing vessel’ for the generation of novel IAV variants. To this end, the emergence of swine influenza viruses must be kept under close surveillance. Herein, we report the isolation and phylogenetic study of a swine IAV, A/swine/Korea/PL01/2012 (swPL01, H3N2 subtype). After screening nasopharyngeal samples from pigs in the Gyeongsangnam-do region of Korea from December 2011 to May 2012, we isolated the swPL01 virus and sequenced its all of 8 genome segments (polymerase basic 2, PB2; polymerase basic 1, PB1; polymerase acidic, PA; hemagglutinin, HA; nucleocapsid protein, NP; neuraminidase, NA; matrix protein, M; and nonstructural protein, NS). The phylogenetic study, analyzed with reference strains registered in the National Center for Biotechnology Information (NCBI) database, indicated that the swPL01 virus was similar to the North American triple-reassortant swine strains and that the HA gene of the swPL01 virus was categorized into swine H3 cluster IV. The swPL01 virus had the M gene of the triple-reassortant swine H3N2 viruses, whereas that of other contemporary strains in Korea was transferred from the 2009 pandemic H1N1 virus. These data suggest the possibility that various swine H3N2 viruses may co-circulate in Korea, which underlines the importance of a sustained surveillance system against swine IAVs. PMID:24523938

Nudiviruses and other large, double-stranded circular DNA viruses of invertebrates: new insights on an old topic.

PubMed

Wang, Yongjie; Jehle, Johannes A

2009-07-01

Nudiviruses (NVs) are a highly diverse group of large, circular dsDNA viruses pathogenic for invertebrates. They have rod-shaped and enveloped nucleocapsids, replicate in the nucleus of infected host cells, and possess interesting biological and molecular properties. The unassigned viral genus Nudivirus has been proposed for classification of nudiviruses. Currently, the nudiviruses comprise five different viruses: the palm rhinoceros beetle virus (Oryctes rhinoceros NV, OrNV), the Hz-1 virus (Heliothis zea NV-1, HzNV-1), the cricket virus (Gryllus bimaculatus NV, GbNV), the corn earworm moth Hz-2 virus (HzNV-2), and the occluded shrimp Monodon Baculovirus reassigned as Penaeus monodon NV (PmNV). Thus far, the genomes of OrNV, GbNV, HzNV-1 and HzNV-2 have been completely sequenced. They vary between 97 and 230kbp in size and encode between 98 and 160 open reading frames (ORFs). All sequenced nudiviruses have 33 ORFs in common. Strikingly, 20 of them are homologous to baculovirus core genes involved in RNA transcription, DNA replication, virion structural components and other functions. Another nine conserved ORFs are likely associated with DNA replication, repair and recombination, and nucleotide metabolism; one is homologous to baculovirus iap-3 gene; two are nudivirus-specific ORFs of unknown function. Interestingly, one nudivirus ORF is similar to polh/gran gene, encoding occlusion body protein matrix and being conserved in Alpha- Beta- and Gammabaculoviruses. Members of nudiviruses are closely related and form a monophyletic group consisting of two sister clades of OrNV/GbNV and HzNVs/PmNV. It is proposed that nudiviruses and baculoviruses derived from a common ancestor and are evolutionarily related to other large DNA viruses such as the insect-specific salivary gland hypertrophy virus (SGHV) and the marine white spot syndrome virus (WSSV).

Hantaviruses and hantavirus pulmonary syndrome, Maranhao, Brazil.

PubMed

Travassos da Rosa, Elizabeth S; Sampaio de Lemos, Elba R; de Almeida Medeiros, Daniele B; Simith, Darlene B; de Souza Pereira, Armando; Elkhoury, Mauro R; Mendes, Wellington S; Vidigal, José R B; de Oliveira, Renata C; D'Andrea, Paulo S; Bonvicino, Cibele R; Cruz, Ana C R; Nunes, Márcio R T; da Costa Vasconcelos, Pedro F

2010-12-01

To confirm circulation of Anajatuba virus in Maranhao, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region.

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.

The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

PubMed

Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

2013-10-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

PubMed

Yoshida, Asuka; Kawabata, Ryoko; Honda, Tomoyuki; Sakai, Kouji; Ami, Yasushi; Sakaguchi, Takemasa; Irie, Takashi

2018-03-01

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-β) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-β-inducing virus. IFN-β induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells. IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

PubMed

Badillo-Vargas, I E; Rotenberg, D; Schneweis, D J; Hiromasa, Y; Tomich, J M; Whitfield, A E

2012-08-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response to Tomato Spotted Wilt Virus Infection

PubMed Central

Badillo-Vargas, I. E.; Rotenberg, D.; Schneweis, D. J.; Hiromasa, Y.; Tomich, J. M.

2012-01-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction. PMID:22696645

Nucleocapsid Protein Annealing of a Primer-Template Enhances (+)-Strand DNA Synthesis and Fidelity by HIV-1 Reverse Transcriptase†

PubMed Central

Kim, Jiae; Roberts, Anne; Yuan, Hua; Xiong, Yong; Anderson, Karen S.

2012-01-01

Human immunodeficiency virus type-1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Using a pre-steady state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3-7 fold) in the rate of incorporation (kpol) by RT as compared to heat annealed primer-template with single nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (Kd) of the nucleotide. These differences in kpol and Kd were not through direct interactions between HIV-1 RT and NCp7. When examining extension by RT, the data suggests that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat annealed primer-template. This enhancement in rate is mediated through interactions with NCp7’s zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV. PMID:22210155

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

PubMed Central

Lederer, Sabine; Lattwein, Erik; Hanke, Merle; Sonnenberg, Karen; Stoecker, Winfried; Lundkvist, Åke; Vaheri, Antti; Vapalahti, Olli; Chan, Paul K. S.; Feldmann, Heinz; Dick, Daryl; Schmidt-Chanasit, Jonas; Padula, Paula; Vial, Pablo A.; Panculescu-Gatej, Raluca; Ceianu, Cornelia; Heyman, Paul; Avšič-Županc, Tatjana; Niedrig, Matthias

2013-01-01

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV). PMID:23593524

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

PubMed Central

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

PubMed

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

Complete genome sequence of a Watermelon silver mottle virus isolate from China.

PubMed

Rao, Xueqin; Wu, Zhuyan; Li, Yuan

2013-06-01

The complete genome of a Watermelon silver mottle virus (WSMoV) (genus Tospovirus, family Bunyaviridae) isolate (WSMoV-GZ) from Guangdong province, China was sequenced. The genomes of WSMoV-GZ contained 3,603, 4,909, and 8,914 nt of small (S), medium (M), and large (L) RNA segments, respectively, and had a genomic organization characteristic of members of the genus Tospovirus. The amino acid sequence of the nucleocapsid (N) protein, S RNA-encoded nonstructural (NSs) protein, M RNA-encoded nonstructural (NSm) protein, Gn/Gc glycoprotein precursor, and RNA-dependent RNA polymerase (RdRp) protein showed 94.3-97.5 % identity with those of other WSMoV isolates. Phylogenetic analysis showed that the N protein of WSMoV-GZ was clustered together with those of the WSMoV isolates. The full sequence of WSMoV-GZ provides a reference genome for comparison with other tospoviruses.

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

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

Evasion of interferon responses by Ebola and Marburg viruses.

PubMed

Basler, Christopher F; Amarasinghe, Gaya K

2009-09-01

The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause frequently lethal viral hemorrhagic fever. These infections induce potent cytokine production, yet these host responses fail to prevent systemic virus replication. Consistent with this, filoviruses have been found to encode proteins VP35 and VP24 that block host interferon (IFN)-alpha/beta production and inhibit signaling downstream of the IFN-alpha/beta and the IFN-gamma receptors, respectively. VP35, which is a component of the viral nucleocapsid complex and plays an essential role in viral RNA synthesis, acts as a pseudosubstrate for the cellular kinases IKK-epsilon and TBK-1, which phosphorylate and activate interferon regulatory factor 3 (IRF-3) and interferon regulatory factor 7 (IRF-7). VP35 also promotes SUMOylation of IRF-7, repressing IFN gene transcription. In addition, VP35 is a dsRNA-binding protein, and mutations that disrupt dsRNA binding impair VP35 IFN-antagonist activity while leaving its RNA replication functions intact. The phenotypes of recombinant EBOV bearing mutant VP35s unable to inhibit IFN-alpha/beta demonstrate that VP35 IFN-antagonist activity is critical for full virulence of these lethal pathogens. The structure of the VP35 dsRNA-binding domain, which has recently become available, is expected to provide insight into how VP35 IFN-antagonist and dsRNA-binding functions are related. The EBOV VP24 protein inhibits IFN signaling through an interaction with select host cell karyopherin-alpha proteins, preventing the nuclear import of otherwise activated STAT1. It remains to be determined to what extent VP24 may also modulate the nuclear import of other host cell factors and to what extent this may influence the outcome of infection. Notably, the Marburg virus VP24 protein does not detectably block STAT1 nuclear import, and, unlike EBOV, MARV infection inhibits STAT1 and STAT2 phosphorylation. Thus, despite their similarities, there are fundamental differences by which

Disruption of Bombyx mori nucleopolyhedrovirus ORF71 (Bm71) results in inefficient budded virus production and decreased virulence in host larvae.

PubMed

Zhang, Min-Juan; Cheng, Ruo-Lin; Lou, Yi-Han; Ye, Wan-Lu; Zhang, Tao; Fan, Xiao-Ying; Fan, Hai-Wei; Zhang, Chuan-Xi

2012-08-01

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects domestic silkworm. BmNPV ORF71 (Bm71) is not a core set gene in baculovirus and shares 92 % amino acid sequence identity with Autographa californica multinucleocapsid NPV ORF88 (Ac88/cg30). Previously, it has been reported that virus lacking Ac88 had no striking phenotypes in cell lines or host larvae. However, the exact role of Bm71 during BmNPV life cycle remains unknown. In the present study, we constructed a Bm71-disrupted (Bm71-D) virus and assessed the effect of the Bm71 disruption on viral replication and viral phenotype throughout the viral life cycle. Results showed that the Bm71-D bacmid could successfully transfect Bm5 cell lines and produce infectious budded virus (BV). But the BV titer was 10- to 100-fold lower than that of the wild-type (WT) virus during infection, and the decreased BV titer was rescued by Bm71 gene repair virus (Bm71-R). A larval bioassay showed that Bm71-D virus took 7.5 h longer than the WT to kill Bombyx mori larvae. Transmission electron microscopy analysis indicated that the Bm71-D virus-infected cells had typical virogenic stroma, bundles of nucleocapsids and polyhedra. Taken together, these results suggest that Bm71 has important implications for determining BV yield and virulence in viral life cycle even though it is not an essential gene for replication of BmNPV.

Small-RNA Deep Sequencing Reveals Arctium tomentosum as a Natural Host of Alstroemeria virus X and a New Putative Emaravirus

PubMed Central

Bi, Yaqi; Tugume, Arthur K.; Valkonen, Jari P. T.

2012-01-01

Background Arctium species (Asteraceae) are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21–24 nucleotides) that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS). Methodology/Principal Findings SRDS was used to detect viruses in the biennial wild plant species Arctium tomentosum (woolly burdock; family Asteraceae) displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX), a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae), or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus—tentatively named as Woolly burdock yellow vein virus—was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested. Conclusions/Significance These results provide novel information about natural virus infections in Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae). Results also revealed a new virus related to the recently emerged Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in plant virology

Hantaviruses and Hantavirus Pulmonary Syndrome, Maranhão, Brazil

PubMed Central

Travassos da Rosa, Elizabeth S.; Sampaio de Lemos, Elba R.; Medeiros, Daniele B. de Almeida; Simith, Darlene B.; Pereira, Armando de Souza; Elkhoury, Mauro R.; Mendes, Wellington S.; Vidigal, José R.B.; de Oliveira, Renata C.; D’Andrea, Paulo S.; Bonvícino, Cibele R.; Cruz, Ana C.R.; Nunes, Márcio R.T.

2010-01-01

To confirm circulation of Anajatuba virus in Maranhão, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region. PMID:21122229

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles

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

Kuznetsov, Yu. G.; Ulbrich, P.; Institute of Molecular Genetics, Czech Academy of Sciences, 166 10 Prague

2007-04-10

Particles of {delta}ProCANC, a fusion of capsid (Canada) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particlesmore » are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, {delta}ProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV {delta}ProCANC.« less

Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.

PubMed

Li, Wenfeng; Desmarets, Lowiese M B; De Gryse, Gaëtan M A; Theuns, Sebastiaan; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans J

2015-09-01

The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.

Cloning the Antibody Response in Humans with Chronic Inflammatory Disease: Immunopanning of Subacute Sclerosing Panencephalitis (SSPE) Brain Sections with Antibody Phage Libraries Prepared from SSPE Brain Enriches for Antibody Recognizing Measles Virus Antigens In Situ

PubMed Central

Owens, Gregory P.; Williamson, R. Anthony; Burgoon, Mark P.; Ghausi, Omar; Burton, Dennis R.; Gilden, Donald H.

2000-01-01

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause. PMID:10627565

Diversity and evolutionary history of lettuce necrotic yellows virus in Australia and New Zealand.

PubMed

Higgins, Colleen M; Chang, Wee-Leong; Khan, Subuhi; Tang, Joe; Elliott, Carol; Dietzgen, Ralf G

2016-02-01

Lettuce necrotic yellows virus (LNYV) is the type member of the genus Cytorhabdovirus, family Rhabdoviridae, and causes a severe disease of lettuce (Lactuca sativa L.). This virus has been described as endemic to Australia and New Zealand, with sporadic reports of a similar virus in Europe. Genetic variability studies of plant-infecting rhabdoviruses are scarce. We have extended a previous study on the variability of the LNYV nucleocapsid gene, comparing sequences from isolates sampled from both Australia and New Zealand, as well as analysing symptom expression on Nicotiana glutinosa. Phylogenetic and BEAST analyses confirm separation of LNYV isolates into two subgroups (I and II) and suggest that subgroup I is slightly older than subgroup II. No correlation was observed between isolate subgroup and disease symptoms on N. glutinosa. The origin of LNYV remains unclear; LNYV may have moved between native and weed hosts within Australia or New Zealand before infecting lettuce or may have appeared as a result of at least two incursions, with the first coinciding with the beginning of European agriculture in the region. The apparent extinction of subgroup I in Australia may have been due to less-efficient dispersal than that which has occurred for subgroup II - possibly a consequence of suboptimal interactions with plant and/or insect hosts. Introduction of subgroup II to New Zealand appears to be more recent. More-detailed epidemiological studies using molecular tools are needed to fully understand how LNYV interacts with its hosts and to determine where the virus originated.

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

PubMed

Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R

2001-04-01

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Structure of acidic pH dengue virus showing the fusogenic glycoprotein trimers.

PubMed

Zhang, Xinzheng; Sheng, Ju; Austin, S Kyle; Hoornweg, Tabitha E; Smit, Jolanda M; Kuhn, Richard J; Diamond, Michael S; Rossmann, Michael G

2015-01-01

Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer. The structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130° to the postfusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" postfusion conformation, thus inhibiting fusion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication.

PubMed

Schuchman, Ryan; Kilianski, Andy; Piper, Amanda; Vancini, Ricardo; Ribeiro, José M C; Sprague, Thomas R; Nasar, Farooq; Boyd, Gabrielle; Hernandez, Raquel; Glaros, Trevor

2018-05-09

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. IMPORTANCE Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

PubMed

Tanchou, V; Gabus, C; Rogemond, V; Darlix, J L

1995-10-06

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Molecular diversity of Rice grassy stunt virus in Vietnam.

PubMed

Ta, Hoang-Anh; Nguyen, Doan-Phuong; Causse, Sandrine; Nguyen, Thanh-Duc; Ngo, Vinh-Vien; Hébrard, Eugénie

2013-04-01

Rice grassy stunt virus (RGSV, Tenuivirus) recently emerged on rice in Vietnam, causing high yield losses during 2006-2009. The genetic diversity of RGSV is poorly documented. In this study, the two genes encoded by each ambisense segment RNA3 and RNA5 of RGSV isolates from six provinces of South Vietnam were sequenced. P3 and Pc3 (RNA3) have unknown function, P5 (RNA5) encodes the putative silencing suppressor, and Pc5 (RNA5) encodes the nucleocapsid protein (N). The sequences of 17 Vietnamese isolates were compared with reference isolates from North and South Philippines. The average nucleotide diversity among the isolates was low. We confirmed a higher variability of RNA3 than RNA5 and Pc3 than P3. No relationships between the genetic diversity and the geographic distribution of RGSV isolates could be ascertained, likely because of the long-distance migration of the insect vector. This data will contribute to a better understanding on the RGSV epidemiology in South Vietnam, a prerequisite for further management of the disease and rice breeding for resistance.

Polypeptide Synthesis in Simian Virus 5-Infected Cells

PubMed Central

Peluso, Richard W.; Lamb, Robert A.; Choppin, Purnell W.

1977-01-01

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F0) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F1 and F2. No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

PubMed

Dietzel, Erik; Schudt, Gordian; Krähling, Verena; Matrosovich, Mikhail; Becker, Stephan

2017-01-15

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of

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

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.

Physicochemical Study of Viral Nanoparticles at the Air/Water Interface.

PubMed

Torres-Salgado, Jose F; Comas-Garcia, Mauricio; Villagrana-Escareño, Maria V; Durán-Meza, Ana L; Ruiz-García, Jaime; Cadena-Nava, Ruben D

2016-07-07

The assembly of most single-stranded RNA (ssRNA) viruses into icosahedral nucleocapsids is a spontaneous process driven by protein-protein and RNA-protein interactions. The precise nature of these interactions results in the assembly of extremely monodisperse and structurally indistinguishable nucleocapsids. In this work, by using a ssRNA plant virus (cowpea chlorotic mottle virus [CCMV]) as a charged nanoparticle we show that the diffusion of these nanoparticles from the bulk solution to the air/water interface is an irreversible adsorption process. By using the Langmuir technique, we measured the diffusion and adsorption of viral nucleocapsids at the air/water interface at different pH conditions. The pH changes, and therefore in the net surface charge of the virions, have a great influence in the diffusion rate from the bulk solution to the air/water interface. Moreover, assembly of mesoscopic and microscopic viral aggregates at this interface depends on the net surface charge of the virions and the surface pressure. By using Brewster's angle microscopy we characterized these structures at the interface. Most common structures observed were clusters of virions and soap-frothlike micron-size structures. Furthermore, the CCMV films were compressed to form monolayers and multilayers from moderate to high surface pressures, respectively. After transferring the films from the air/water interface onto mica by using the Langmuir-Blodgett technique, their morphology was characterized by atomic force microscopy. These viral monolayers showed closed-packing nano- and microscopic arrangements.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico

PubMed Central

Ramírez-Hernández, Dolores G.; Lara-Padilla, Eleazar; Zárate-Segura, Paola

2016-01-01

Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

PubMed

Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

2016-01-01

Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

PubMed

Nelson, Emily V; Schmidt, Kristina M; Deflubé, Laure R; Doğanay, Sultan; Banadyga, Logan; Olejnik, Judith; Hume, Adam J; Ryabchikova, Elena; Ebihara, Hideki; Kedersha, Nancy; Ha, Taekjip; Mühlberger, Elke

2016-08-15

A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein.

PubMed

Gely, Stéphane; Lowry, David F; Bernard, Cédric; Jensen, Malene R; Blackledge, Martin; Costanzo, Stéphanie; Bourhis, Jean-Marie; Darbon, Hervé; Daughdrill, Gary; Longhi, Sonia

2010-01-01

In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.

Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

PubMed Central

Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

2015-01-01

ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the

[Microbiological surveillance: viral hemorrhagic fever in Central African Republic: current serological data in man].

PubMed

Nakounné, E; Selekon, B; Morvan, J

2000-01-01

An investigation was conducted between 1994 and 1997 in forested areas of the Central African Republic (CAR) to determine the seroprevalence of IgG antibodies against several haemorrhagic fever viruses present in the region. Sera were obtained from 1762 individuals in two groups (Pygmy and Bantu locuted populations) living in 4 forested areas in the south of the country. Sera were tested for IgG antibodies against Ebola, Marburg, Rift Valley fever (RVF), Yellow fever (YF) and Hantaviruses by enzyme immunoassay (EIA), and against Lassa virus by immunofluorescent assay. The prevalence of IgG antibodies was 5.9% for Ebola, 2% for Marburg, 6.9% pour RVF, 6.5% for YF, 2% for Hantaan. No antibodies were detected against Lassa, Seoul, Puumala and Thottapalayam viruses. No IgM antibodies were detected against RVF and YF viruses. The distribution of antibodies appears to be related to tropical rain forest areas. This study indicates that several haemorrhagic fever viruses are endemic in forested areas of the CAR and could emerge due to environmental modification.

Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

PubMed Central

Dinh, Phat X.; Das, Anshuman; Franco, Rodrigo

2013-01-01

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the family of hnRNPs and was recently shown in a genome-wide small interfering RNA (siRNA) screen to support vesicular stomatitis virus (VSV) growth. To decipher the role of hnRNP K in VSV infection, we conducted studies which suggest that the protein is required for VSV spreading. Virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnRNP K, whereas viral genome transcription and replication were reduced slightly. These results indicate that hnRNP K is likely involved in virus assembly and/or release from infected cells. Further studies showed that hnRNP K suppresses apoptosis of virus-infected cells, resulting in increased cell survival during VSV infection. The increased survival of the infected cells was found to be due to the suppression of proapoptotic proteins such as Bcl-XS and Bik in a cell-type-dependent manner. Additionally, depletion of hnRNP K resulted in not only significantly increased levels of T-cell-restricted intracellular antigen 1 (TIA1) but also switching of the expression of the two isoforms of the protein (TIA1a and TIA1b), both of which inhibited VSV replication. hnRNP K was also found to support expression of several cellular proteins known to be required for VSV infection. Overall, our studies demonstrate hnRNP K to be a multifunctional protein that supports VSV infection via its role(s) in suppressing apoptosis of infected cells, inhibiting the expression of antiviral proteins, and maintaining the expression of proteins required for the virus. PMID:23843646

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

PubMed

Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

1990-12-05

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Isolation and serological differentiation of a herpesvirus from bobwhite quail (Colinus virginianus, L. 1758).

PubMed

Kaleta, E F; Marschall, H J; Glünder, G; Stiburek, B

1980-01-01

An infectious agent was isolated from the liver of bobwhite quails (Colinus virginianus, L. 1758). The agent was sensitive to chloroform and its multiplication was inhibited by 5-iodine-2-deoxy-uridine. It passed filters with a pore diameter of 220 nm and more but not 100 nm filters. Electron microscopic examination revealed numerous nucleocapsids with hollow capsomeres and few enveloped particles in the supernatant fluids of infected cultures. The nucleocapsids were calculated to have 162 capsomeres on their surface. Using the plaque reduction method for neutralization tests no serological cross reactions could be detected between the quail isolate and sera against Marek's disease virus, turkey herpes-virus (HV), duck enteritis HV, infectious laryngotracheitis HV, amazon parrot HV, great horned owl HV, eagle owl HV, snowy owl HV, falcon HV, pigeon HV, Lake Victoria Cormorant HV, and stork HV. The isolate from bobwhite quail did only cross-react with antiserum against crane HV. It is concluded that the isolated virus is a member of the avian herpesvirus group and it is proposed to tentatively term it herpesvirus colinum (from Colinus virginianus = bobwhite quail).

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

PubMed

Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

1997-05-02

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

Transformation of primary chick embryo fibroblasts by Marek's disease virus.

PubMed

Buranathai, C; Rodriguez, J; Grose, C

1997-12-08

Marek's disease virus (MDV) is an alphaherpesvirus, which can mediate the malignant transformation of lymphocytes to form lymphomas in chickens. In this study, we demonstrate that MDV can transform primary chick embryo fibroblasts (CEF). The cell line derived from primary CEF infected with the GA strain of MDV was called CEM(MDV). The fibroblast nature of CEM(MDV) was verified by absence of cytokeratin type II. The CEM(MDV) phenotype differed from either primary CEF or MDV-infected CEF. CEM(MDV) were extensively vacuolated, with unusual multilamellar structures in the cytoplasm, The nuclei were considerably larger than those in primary CEF and were uniformly positive for proliferating cell nuclear antigen. The cell line was subcultured for more than 10 generations; however, CEM(MDV) did not support a fully productive MDV infection, because complete nucleocapsids were not detected and infectivity assays showed that cell line produced no infectious virus. PCR analyses demonstrated that this cell line carried both polypeptide 38 (pp38) and Meq DNA, MDV-specific genes associated with transformation. In addition, examination by laser scanning confocal microscopy revealed that CEM(MDV) constitutively produced MDV MEQ protein in nuclei and pp38 as well as glycoprotein B in the cytoplasm and on the plasma membrane. Growth in soft agar assay demonstrated that CEM(MDV) formed colonies, similar to HeLa and human melanoma cells. Retroviral insertion was not detected in DNA from the CEM(MDV) line.

Research in drug development against viral diseases of military importance (biological testing). Volume 2. Final report, 15 November 1985-31 January 1991

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

Shannon, W.M.; Arnett, G.; Brazier, A.D.

1991-03-01

The purpose of this program is to evaluate the efficacy of candidate antiviral compounds against a spectrum of viruses of military importance. This program involves (a) primary testing of chemical compounds and natural products for antiviral efficacy in vitro using standard CPE-inhibition assays, (b) primary testing of compounds for antiviral efficacy in vivo in animal model systems, and (c) secondary evaluation of the active candidate antiviral compounds. The target viruses for in vitro testing are Vaccinia Virus (VV), Adenovirus (AD2), Vesicular Stomatitis Virus (VSV), Punta Toro Virus (PT), Sandfly fever Virus (SF), Yellow Fever Virus (YF), Venezuelan Equine Encephalomyelitis Virusmore » (VE), Japanese Encephalitis Virus, Pichinde Virus (PIC), Hantaan Virus (HTN), and Human Immunodeficiency Virus (HIV). The in vivo systems are Pichinde Virus infection of hamsters, Venezuelan Equine Encephalomyelitis Virus, Japanese Encephalitis Virus and Vaccinia virus infections of mice. Approximately 10,000 compounds have been received for in vitro evaluation and over 66,000 assays have been performed on this contract. Compounds have been identified in nearly all virus systems that have confirmed antiviral activity equal or exceeding that of the various positive control compounds (ribavirin, selenazofurin, carbocyclic-3-aza-adenosine, adenosine dialdehyde, Ara-A, ddC and AZT). Many of these compounds represent potent and selective new antiviral agents.« less

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

The Continuing Evolution of H5N1 and H9N2 Influenza Viruses in Bangladesh Between 2013 and 2014.

PubMed

Marinova-Petkova, Atanaska; Shanmuganatham, Karthik; Feeroz, Mohammed M; Jones-Engel, Lisa; Hasan, M Kamrul; Akhtar, Sharmin; Turner, Jasmine; Walker, David; Seiler, Patrick; Franks, John; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-05-01

In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra-and interclade reassortant; its HA, polymerase basic 1 (PB1), polymerase (PA), and nonstructural (NS) genes come from subclade 2.3.2.1a; the polymerase basic 2 (PB2) comes from subclade 2.3.2.1c; and the NA, nucleocapsid protein (NP), and matrix (M) gene from clade 2.3.4.2. The H9N2 influenza viruses cocirculating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from an HPAI H7N3 virus previously isolated in Pakistan. Despite frequent coinfection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried seven genes from the local HPAI H5N1 lineage and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although the live birds we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans.

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

PubMed

Tóth, F D; Aboagye-Mathiesen, G; Szabó, J; Liu, X; Mosborg-Petersen, P; Kiss, J; Hager, H; Zdravkovic, M; Andirkó, I; Aranyosi, J

1995-12-01

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Intracellular Virus-Specific Structures and RNAs in Oncornavirus-Producing Human Cells

PubMed Central

Bukrinskaya, A. G.; Miller, G. G.; Lebedeva, E. N.; Zhdanov, V. M.

1974-01-01

Two kinds of virus-specific structures were isolated from the cytoplasm of Detroit-6 and human amnion cells producing oncornavirus-like particles. These structures represented A particles with the diameter of 70 to 80 nm and aggregated strands of nucleocapsids with the diameter of 3 and 6 nm. The structures were separated from cellular contaminants by isopycnic banding in linear sucrose gradients and subsequently further purified by sedimentation in velocity sucrose gradients. Their sedimentation coefficient was 250 and 150S, respectively. Both structures contain 60, 45, and 35S RNA species, and 150S structures also contained 20S RNA. The 35 and 20S RNA from the 150S structure formed hybrids with DNA enzymatically synthesized on extracellular virions. The structures displayed endogeneous polymerase activity, DNA product of the reaction being predominantly associated with 60S RNA. No 70S RNA was found in the cell structures of various densities. Also, the virions purified from tissue culture fluid contained 70S RNA. These findings are consistent with those on extracellular maturation of oncornavirus RNA. Images PMID:4810779

Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

PubMed

Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

2005-05-20

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

Targeting a Dark Excited State of HIV-1 Nucleocapsid by Antiretroviral Thioesters Revealed by NMR Spectroscopy.

PubMed

Deshmukh, Lalit; Tugarinov, Vitali; Appella, Daniel H; Clore, G Marius

2018-03-01

HIV-1 nucleocapsid (NCp7) is a two Cys 2 HisCys zinc knuckle (N-Zn and C-Zn) protein that plays a key role in viral replication. NCp7 conformational dynamics is characterized by NMR relaxation dispersion and chemical exchange saturation transfer measurements. While the N-Zn knuckle is conformationally stable, the C-Zn knuckle interconverts on the millisecond timescale between the major state, in which the zinc is coordinated by three cysteines and a histidine, and two folded minor species (with populations around 1 %) in which one of the coordination bonds (Cys413-Sγ-Zn or His421-Nϵ2-Zn) is hydrolyzed. These findings explain why antiretroviral thioesters specifically disrupt the C-Zn knuckle by initial acylation of Cys413, and show that transient, sparsely-populated ("dark"), excited states of proteins can present effective targets for rational drug design. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

PubMed

Gamba, Elia; Mori, Mattia; Kovalenko, Lesia; Giannini, Alessia; Sosic, Alice; Saladini, Francesco; Fabris, Dan; Mély, Yves; Gatto, Barbara; Botta, Maurizio

2018-02-10

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Genetic characterization of measles virus in the Philippines, 2008-2011.

PubMed

Centeno, Rex; Fuji, Naoko; Okamoto, Michiko; Dapat, Clyde; Saito, Mariko; Tandoc, Amado; Lupisan, Socorro; Oshitani, Hitoshi

2015-06-03

Large outbreaks of measles occurred in the Philippines in 2010 and 2011. Genetic analysis was performed to identify the genotype of measles virus (MeV) that was responsible for the large outbreaks. A total of 114 representative MeVs that were detected in the Philippines from 2008 to 2011 were analyzed by sequencing the C-terminal region of nucleocapsid (N) gene and partial hemagglutinin (H) gene and by inferring the phylogenetic trees. Genetic analysis showed that genotype D9 was the predominant circulating strain during the 4-year study period. Genotype D9 was detected in 23 samples (92%) by N gene sequencing and 93 samples (94%) by H gene analysis. Sporadic cases of genotype G3 MeV were identified in 2 samples (8%) by N gene sequencing and 6 samples (6%) by H gene analysis. Genotype G3 MeV was detected mainly in Panay Island in 2009 and 2010. Molecular clock analysis of N gene showed that the recent genotype D9 viruses that caused the big outbreaks in 2010 and 2011 diverged from a common ancestor in 2005 in one of the neighboring Southeast Asian countries, where D9 was endemic. These big outbreaks of measles resulted in a spillover and were associated with genotype D9 MeV importation to Japan and the USA. Genotype D9 MeV became endemic and caused two big outbreaks in the Philippines in 2010 and 2011. Genotype G3 MeV was detected sporadically with limited geographic distribution. This study highlights the importance of genetic analysis not only in helping with the assessment of measles elimination program in the country but also in elucidating the transmission dynamics of measles virus.

A glycoprotein subunit vaccine elicits a strong Rift Valley fever virus neutralizing antibody response in sheep.

PubMed

Faburay, Bonto; Lebedev, Maxim; McVey, D Scott; Wilson, William; Morozov, Igor; Young, Alan; Richt, Juergen A

2014-10-01

Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species.

A Glycoprotein Subunit Vaccine Elicits a Strong Rift Valley Fever Virus Neutralizing Antibody Response in Sheep

PubMed Central

Lebedev, Maxim; McVey, D. Scott; Wilson, William; Morozov, Igor; Young, Alan

2014-01-01

Abstract Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species. PMID:25325319

Persistent measles virus infection of the intestine: confirmation by immunogold electron microscopy.

PubMed Central

Lewin, J; Dhillon, A P; Sim, R; Mazure, G; Pounder, R E; Wakefield, A J

1995-01-01

This study sought to investigate persistent measles virus infection of the intestine: a novel protocol for immunogold electron microscopy was developed using a polyclonal anti-measles nucleoprotein antibody on reprocessed, formalin fixed paraffin wax embedded tissue sections. Antibody binding was detected using both immunoperoxidase and light microscopy on tissue sections, and 10 nm gold conjugated secondary antibody and electron microscopy on ultrathin sections. The techniques were validated using both measles infected vero cells and human tissues with established measles infection: these included brain affected by subacute sclerosing panencephalitis and acute measles appendicitis. The technique was applied subsequently to six untreated cases of granulomatous Crohn's disease, and two cases of ileocaecal tuberculosis, a granulomatous control. Mumps primary antibody--applied to both mumps infected vero cells, and measles infected vero cells and tissues studied by immunoperoxidase, and measles antibody on mumps infected cells studied by immunoperoxidase and immunogold--were used as specificity controls: the primary antibodies identified their respective target antigen and there was no antibody cross reactivity. Measles virus nucleocapsids labelled with gold conjugated antibody in both infected cells and tissues, including foci of granulomatous inflammation in five of six cases of Crohn's disease: in the fifth case, the granuloma could not be identified in ultrathin section. In one of the tuberculosis cases, a low level of signal was noted while the second case was negative. Labelling adopted a characteristic pattern in all infected tissues, strengthening the specificity of these findings. This study provides the first direct confirmation of persistent measles virus infection of the intestine. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7737565

Characterization of Coffee ringspot virus-Lavras: A model for an emerging threat to coffee production and quality

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

Ramalho, T.O.; Figueira, A.R.; Sotero, A.J.

2014-09-15

The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a membermore » of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. - Highlights: • We report genome sequence determination for Coffee ringspot virus (CoRSV). • CoRSV should be considered a member of the proposed Dichorhavirus genus. • We report temperature-dependent systemic infection of an initially local lesion host. • We report in planta protein and localization data for five CoRSV proteins. • In silico predictions of the CoRSV proteins were validated using in vivo assays.« less

Hepatitis B virus replication

PubMed Central

Beck, Juergen; Nassal, Michael

2007-01-01

Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ε RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development. PMID:17206754

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly.

PubMed

Wu, Shuo; Zhao, Qiong; Zhang, Pinghu; Kulp, John; Hu, Lydia; Hwang, Nicky; Zhang, Jiming; Block, Timothy M; Xu, Xiaodong; Du, Yanming; Chang, Jinhong; Guo, Ju-Tao

2017-08-15

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV. IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several

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

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses

PubMed Central

Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A.; Maines, Taronna R.

2016-01-01

ABSTRACT A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary

The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions ▿ †

PubMed Central

Kostyuchenko, Victor A.; Jakana, Joanita; Liu, Xiangan; Haddow, Andrew D.; Aung, Myint; Weaver, Scott C.; Chiu, Wah; Lok, Shee-Mei

2011-01-01

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design. PMID:21752915

Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

PubMed

Zhang, Min-Juan; Tian, Cai-Hong; Fan, Xiao-Ying; Lou, Yi-Han; Cheng, Ruo-Lin; Zhang, Chuan-Xi

2012-07-01

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

PubMed

Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

2008-06-22

Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.

One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

PubMed

Park, Sun-Whan; Lee, Ye-Ji; Lee, Won-Ja; Jee, Youngmee; Choi, WooYoung

2016-06-01

Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA. We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.

S-phase arrest after vincristine treatment may promote hepatitis B virus replication

PubMed Central

Xu, Lei; Tu, Zeng; Xu, Ge; Hu, Jie-Li; Cai, Xue-Fei; Zhan, Xing-Xing; Wang, Yu-Wei; Huang, Yuan; Chen, Juan; Huang, Ai-Long

2015-01-01

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication. PMID:25663769

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

PubMed

Wilson, M E; Consigli, R A

1985-06-01

A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Molecular epidemiology reveals emergence of a virulent infectious hematopoietic necrosis (IHN) virus strain in wild salmon and its transmission to hatchery fish

USGS Publications Warehouse

Anderson, Eric D.; Engelking, H. Mark; Emmenegger, Eveline J.; Kurath, Gael

2000-01-01

Infectious hematopoietic necrosis virus (IHNV) has been known to be a significant salmonid pathogen in the Pacific Northwest of North America for decades. The goal of this study was to characterize the IHNV genetic heterogeneity and viral traffic over time at a study site in the Deschutes River watershed in Oregon, with an emphasis on the epidemiology of IHNV types causing epidemics in wild kokanee Oncorhynchus nerkabetween 1991 and 1995. The study site included kokanee spawning grounds in the Metolius River and Lake Billy Chinook downstream, in which the IHNV epidemics occurred in 2- and 3-year-old kokanee, and the Round Butte Fish Hatchery at the outflow of the lake. Forty-two IHNV isolates collected from this area between 1975 and 1995 were characterized on a genetic basis by ribonuclease (RNase) protection fingerprint analyses of the virus nucleocapsid, glycoprotein, and nonvirion genes. Analysis of the 16 identified composite haplotypes suggested that both virus evolution and introduction of new IHNV strains contributed to the genetic diversity observed. The results indicated that the 1991–1995 epidemics in kokanee from Lake Billy Chinook were due to a newly introduced IHNV type that was first detected in spawning adult kokanee in 1988 and that this virus type was transmitted from the wild kokanee to hatchery fish downstream in 1991. Twelve IHNV haplotypes were found at Round Butte Fish Hatchery, indicating a series of virus displacement events during the 20-year period examined. This work shows that IHNV traffic can be much more complex than was previously recognized, and the results have implications for fisheries management at the hatchery and throughout the watershed.

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

PubMed

Qi, Yonghe; Gao, Zhenchao; Xu, Guangwei; Peng, Bo; Liu, Chenxuan; Yan, Huan; Yao, Qiyan; Sun, Guoliang; Liu, Yang; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

2016-10-01

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2012-01-01

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome. PMID:22745685

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

USGS Publications Warehouse

Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

2010-01-01

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Wild-Type Measles Viruses with Non-Standard Genome Lengths

PubMed Central

Bankamp, Bettina; Liu, Chunyu; Rivailler, Pierre; Bera, Jayati; Shrivastava, Susmita; Kirkness, Ewen F.; Bellini, William J.; Rota, Paul A.

2014-01-01

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible. PMID:24748123

A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

PubMed

Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

2016-06-01

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed

Bieth, E; Gabus, C; Darlix, J L

1990-01-11

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

PubMed

Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

2016-12-15

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage

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.

Family Ophioviridae: classification and features

USDA-ARS?s Scientific Manuscript database

The Ophioviridae is a family of filamentous plant viruses, with a single stranded negative, and possibly ambisense, RNA genome of 11.3–12.5 kb divided into 3–4 segments, each encapsidated by a single coat protein. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two d...

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

Treesearch

James McNeil; Diana Cox-Foster; James Slavicek; Kelli Hoover

2010-01-01

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its...

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

PubMed

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

Adler hantavirus, a new genetic variant of Tula virus identified in Major's pine voles (Microtus majori) sampled in southern European Russia.

PubMed

Tkachenko, Evgeniy A; Witkowski, Peter T; Radosa, Lukas; Dzagurova, Tamara K; Okulova, Nataliya M; Yunicheva, Yulia V; Vasilenko, Ludmila; Morozov, Vyacheslav G; Malkin, Gennadiy A; Krüger, Detlev H; Klempa, Boris

2015-01-01

Although at least 30 novel hantaviruses have been recently discovered in novel hosts such as shrews, moles and even bats, hantaviruses (family Bunyaviridae, genus Hantavirus) are primarily known as rodent-borne human pathogens. Here we report on identification of a novel hantavirus variant associated with a rodent host, Major's pine vole (Microtus majori). Altogether 36 hantavirus PCR-positive Major's pine voles were identified in the Krasnodar region of southern European Russia within the years 2008-2011. Initial partial L-segment sequence analysis revealed novel hantavirus sequences. Moreover, we found a single common vole (Microtusarvalis) infected with Tula virus (TULV). Complete S- and M-segment coding sequences were determined from 11 Major's pine voles originating from 8 trapping sites and subjected to phylogenetic analyses. The data obtained show that Major's pine vole is a newly recognized hantavirus reservoir host. The newfound virus, provisionally called Adler hantavirus (ADLV), is closely related to TULV. Based on amino acid differences to TULV (5.6-8.2% for nucleocapsid protein, 9.4-9.5% for glycoprotein precursor) we propose to consider ADLV as a genotype of TULV. Occurrence of ADLV and TULV in the same region suggests that ADLV is not only a geographical variant of TULV but a host-specific genotype. High intra-cluster nucleotide sequence variability (up to 18%) and geographic clustering indicate long-term presence of the virus in this region. Copyright © 2014. Published by Elsevier B.V.

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

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

PubMed

Globig, A; Fereidouni, S R; Harder, T C; Grund, C; Beer, M; Mettenleiter, T C; Starick, E

2013-10-01

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds. © 2012 Blackwell Verlag GmbH.

Community-Based Prevalence Profile of Arboviral, Rickettsial, and Hantaan-Like Viral Antibody in the Nile River Delta of Egypt

DTIC Science & Technology

1993-01-01

presentation. Susan Hibbsfor assisting in the preparation of the data for presen- cates that the antibody response is strengthened tation. Dr. Atef Soliman...virus: a newly 630-633. recognized arthropod transmitted virus. AIntJ 3. Scott RM. Feinsod FNM, Allam IH. Ksiazek TG. Trop Med H * g 4: 844-862. Peters

Family Ophioviridae

USDA-ARS?s Scientific Manuscript database

The Ophioviridae is a family of filamentous plant viruses, with a single stranded negative, and possibly ambisense, RNA genome of 11.3–12.5 kb in length divided into 3–4 segments, each separately encapsidated by a single coat protein (43–50 kDa). Virions are naked filamentous nucleocapsids, in shape...

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

PubMed Central

Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

2016-01-01

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed Central

Bieth, E; Gabus, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12. Images PMID:2155394

Hantaviruses induce cell type- and viral species-specific host microRNA expression signatures

PubMed Central

Shin, Ok Sarah; Kumar, Mukesh; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The mechanisms of hantavirus-induced modulation of host cellular immunity remain poorly understood. Recently, microRNAs (miRNAs) have emerged as a class of essential regulators of host immune response genes. To ascertain if differential host miRNA expression toward representative hantavirus species correlated with immune response genes, miRNA expression profiles were analyzed in human endothelial cells, macrophages and epithelial cells infected with pathogenic and nonpathogenic rodent- and shrew-borne hantaviruses. Distinct miRNA expression profiles were observed in a cell type- and viral species-specific pattern. A subset of miRNAs, including miR-151-5p and miR-1973, were differentially expressed between Hantaan virus and Prospect Hill virus. Pathway analyses confirmed that the targets of selected miRNAs were associated with inflammatory responses and innate immune receptor-mediated signaling pathways. Our data suggest that differential immune responses following hantavirus infection may be regulated in part by cellular miRNA through dysregulation of genes critical to the inflammatory process. PMID:24074584

Fugong virus, a novel hantavirus harbored by the small oriental vole (Eothenomys eleusis) in China.

PubMed

Ge, Xing-Yi; Yang, Wei-Hong; Pan, Hong; Zhou, Ji-Hua; Han, Xi; Zhu, Guang-Jian; Desmond, James S; Daszak, Peter; Shi, Zheng-Li; Zhang, Yun-Zhi

2016-02-16

Rodents are natural reservoirs of hantaviruses, which cause two disease types: hemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in North America. Hantaviruses related human cases have been observed throughout Asia, Europe, Africa, and North America. To date, 23 distinct species of hantaviruses, hosted by reservoir, have been identified. However, the diversity and number of hantaviruses are likely underestimated in China, and hantavirus species that cause disease in many regions, including Yunnan province, are unknown. In August 2012, we collected tissue samples from 189 captured animals, including 15 species belonging to 10 genera, 5 families, and 4 orders in Fugong county, Yunnan province, China. Seven species were positive for hantavirus: Eothenomys eleusis (42/94), Apodemus peninsulae (3/25), Niviventer eha (3/27), Cryptotis montivaga (2/8), Anourosorex squamipes (1/1), Sorex araneus (1/1), and Mustela sibirica (1/2). We characterized one full-length genomic sequence of the virus (named fugong virus, FUGV) from a small oriental vole (Eothenomys eleusis). The full-length sequences of the small, medium, and large segments of FUGV were 1813, 3630, and 6531 nt, respectively. FUGV was most closely related to hantavirus LX309, a previously reported species detected in the red-backed vole in Luxi county, Yunnan province, China. However, the amino acid sequences of nucleocapsid (N), glycoprotein (G), and large protein (L) were highly divergent from those of Hantavirus LX309, with amino acid differences of 11.2, 15.3, and 12.7 %, respectively. In phylogenetic trees, FUGV clustered in the lineage corresponding to hantaviruses carried by rodents in the subfamily Arvicolinae. High prevalence of hantavirus infection in small mammals was found in Fugong county, Yunnan province, China. A novel hantavirus species FUGV was identified from the small oriental vole. This virus is phylogenetic clustering with another hantavirus LX309, but shows

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

PubMed Central

Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

2016-01-01

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA.

PubMed

Andersson, Cecilia; Henriksson, Sara; Magnusson, Karl-Eric; Nilsson, Mats; Mirazimi, Ali

2012-05-10

Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly. Copyright © 2012 Elsevier Inc. All rights reserved.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

PubMed

Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

2017-09-01

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B

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

Thuy, Nguyen Thanh, E-mail: ngtthuy02@yahoo.com; Huy, Tran Quang, E-mail: huytq@nihe.org.vn; Nga, Phan Thi

We describe the ultrastructure of the NamDinh virus (NDiV), a new member of the order Nidovirales grown in the C6/36 mosquito cell line. Uninfected and NDiV-infected cells were investigated by electron microscopy 24–48 h after infection. The results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. Mature virions of NDiV were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. Free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. The sizemore » of the round-shaped particles with a complete internal structure was 80 nm in diameter. This is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus NDiV, a missing evolutionary link in the emergence of the viruses with the largest RNA genomes. - Highlights: • NamDinh virus (NDiV), a new member of the order Nidovirales was tested in cultured cell line. • The morphogenesis and ultrastructure of NDiV were investigated by electron microscopy. • The viral nucleocapsid-like particles clustered and scattered in the cytoplasm. • NDiVs were released as budding particles on the cell surface. • The size of the viral particles with a complete internal structure was 80 nm in diameter.« less

Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs

PubMed Central

Fischer, Kerstin; Diederich, Sandra; Smith, Greg; Reiche, Sven; Pinho dos Reis, Vinicius; Stroh, Eileen; Groschup, Martin H.; Weingartl, Hana M.

2018-01-01

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990’s causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses. PMID:29708971

Structural Studies on Varicella Zoster Virus

DTIC Science & Technology

1990-08-20

TABLE OF CONTENTS INTRODUCTION 1 The VZV Genome , 8 VZV Proteins 12 VZV Transcription 14 The Structure of HSV 15 Herpesvirus Expression 16...VZV virion . . . 4 Figure 2. A drawing of the VZV nucleocapsid 6 Figure 3. A comparison of the structure of the genomes of HSV - 1 and VZV 9 Figure 4...VZV, and purified VZ virions probed with antibody against VZV IE62 (the HSV - 1 ICP4 equivalent) . . . 154 xii Figure 48. Autoradiograph of VZV IE62

Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

Cancer.gov

During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the

Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

PubMed

Kalaiselvan, Sagadevan; Sankar, Sathish; Ramamurthy, Mageshbabu; Ghosh, Asit Ranjan; Nandagopal, Balaji; Sridharan, Gopalan

2017-08-01

Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 118: 2320-2324, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

PubMed

Müller, Barbara; Anders, Maria; Reinstein, Jochen

2014-01-01

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro.

PubMed

Murillo, Juliana Londoño; Cabral, Aline Diniz; Uehara, Mabel; da Silva, Viviam Moura; Dos Santos, Juliete Vitorino; Muniz, João Renato Carvalho; Estrozi, Leandro Farias; Fenel, Daphna; Garcia, Wanius; Sperança, Márcia Aparecida

2018-06-01

Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.

Antiviral activity of a synthesized shikonin ester against influenza A (H1N1) virus and insights into its mechanism.

PubMed

Zhang, Yahan; Han, Hongwei; Qiu, Hanyue; Lin, Hongyan; Yu, Lugang; Zhu, Wanzhan; Qi, Jinliang; Yang, Rongwu; Pang, Yanjun; Wang, Xiaoming; Lu, Guihua; Yang, Yonghua

2017-09-01

This study aimed to examine the antiviral effects of shikonin ester ((R)-1-(5, 8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl3-(1H- indol-3-yl) propanoate (PMM-034) against influenza A (H1N1) virus. We investigated PMM-034 anti-H1N1 activity and its effect on caspase 3 gene expression during cellular apoptosis after influenza virus infection in vitro. Neuraminidase (NA) inhibition was assessed in comparison with oseltamivir in the influenza virus standard strains A/PR/8/34 to understand the viral mechanism. MDCK and A549 cells were used to investigate influenza viral infection and the structure-activity relationship between PMM-034 and NA was evaluated by pharmacophore-based docking modeling. The production of viral protein was tested by western blot. A/PR/8/34 induced cell inhibition but this was reduced by PMM-034 to 16μg/mL and this showed a selective index of 10mM. PMM-034 inhibited NA in a dose dependent manner, similar to oseltamivir inhibition. A sharp decrease in viral nucleocapsid protein mRNA was observed in infected cells after treatment with PMM-034. Apoptosis of infected A459 cells was inhibited by PMM-034 with decreased caspase 3 levels. ARG 118, ARG 152, ARG 371 and GLU 227 in the binding pocket of NA bound to PMM-034 in the docking model. Taken together, these results suggest PMM-034 shikonin ester blocked H1N1 infection and might be a potential anti-H1N1 drug. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Differential Requirements in Endocytic Trafficking for Penetration of Dengue Virus

PubMed Central

Acosta, Eliana G.; Castilla, Viviana; Damonte, Elsa B.

2012-01-01

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection. PMID:22970315

A measles outbreak in Sindh, Pakistan caused by a genotype B3 virus.

PubMed

Zaidi, Syed Sohail Zahoor; Hameed, Abdul; Ali, Naeem; Umair, Massab; Alam, Muhammad Masroor; Rana, Muhammad Suleman; Sharif, Salmaan; Aamir, Uzma Bashir; Shaukat, Shahzad; Angez, Mehar; Khurshid, Adnan; Akhtar, Ribqa; Mehmood, Nayab; Badar, Nazish

2017-12-01

Measles continues to be a major public health issue causing substantial outbreaks worldwide, mostly affecting young children. Molecular analysis of measles viruses provides important information on outbreak linkages and transmission pathways that can be helpful towards implementation of appropriate control programs. In Pakistan, the control of measles is still tenuous, and progress towards elimination has been irregular and challenging. In the 2013 measles outbreak we received 4,682 sera collected from suspected patients in 23 districts across Sindh. A total of 3,283 samples were confirmed measles positive using IgM ELISA with the highest infection rate in children aged 1-12 months. Males were more affected than females and a visible peak was observed from January to April. Among the 3,283 cases, 59.1% were unvaccinated, 29.6% had received 1 dose and 10.3% had received 2 doses of measles vaccine while 0.85% had an unknown vaccination status. For genotype detection and phylogenetic analysis, 60 throat swab samples were collected from suspected patients below 15 years of age in eight districts of Sindh province. Forty four (73%; 44/60) throat swab samples were successfully genotyped using RT-PCR. Phylogenetic analyses based on partial sequences of the nucleocapsid protein gene revealed that all Pakistani measles virus strains belonged to genotype B3 and were closely related to those isolated from neighboring countries such as Iran, Afghanistan (99.1-100%) and India with 98.6 - 99.6% nucleotide homology. This is the first report on the phylogenetic analysis of measles B3 genotype strains from Pakistan and highlights the need for strengthening the surveillance systems and improving immunization coverage across the country.

Evaluation of Substituted 1,2,3-Dithiazoles as Inhibitors of the Feline Immunodeficiency Virus (FIV) Nucleocapsid Protein via a Proposed Zinc Ejection Mechanism.

PubMed

Asquith, Christopher R M; Konstantinova, Lidia S; Laitinen, Tuomo; Meli, Marina L; Poso, Antti; Rakitin, Oleg A; Hofmann-Lehmann, Regina; Hilton, Stephen T

2016-10-06

A diverse library of 5-thieno-, 5-oxo-, and 5-imino-1,2,3-dithiazole derivatives was synthesized and evaluated for efficacy against the feline immunodeficiency virus (FIV) as a model for HIV in cells. Several diverse compounds from this series displayed nanomolar activity and low toxicity, representing a potential new class of compounds for the treatment of FIV and HIV. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

PubMed

Giles, J C; Johnson, W; Jones, G; Heuer, C; Dunowska, M

2018-07-01

To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand. Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000-2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450 nm (OD 450 ) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus. Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD 450 ≥0.41 for samples positive, and OD 450 <0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196-0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001). The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to

Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress

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

Lymberopoulos, Maria H.; Bourget, Amelie; Abdeljelil, Nawel Ben

2011-04-10

UL24 of herpes simplex virus 1 (HSV-1) is widely conserved within the Herpesviridae family. Herein, we tested the hypothesis that UL24, which we have previously shown to induce the redistribution of nucleolin, also affects the localization of the nucleolar protein B23. We found that HSV-1-induced dispersal of B23 was dependent on UL24. The conserved N-terminal portion of UL24 was sufficient to induce the redistribution of B23 in transient transfection assays. Mutational analysis revealed that the endonuclease motif of UL24 was important for B23 dispersal in both transfected and infected cells. Nucleolar protein relocalization during HSV-1 infection was also observed inmore » non-immortalized cells. Analysis of infected cells by electron microscopy revealed a decrease in the ratio of cytoplasmic versus nuclear viral particles in cells infected with a UL24-deficient strain compared to KOS-infected cells. Our results suggest that UL24 promotes nuclear egress of nucleocapsids during HSV-1 infection, possibly though effects on nucleoli.« less

Isolation and Molecular Characterization of Novel Infectious Bronchitis Virus Variants from Vaccinated Broiler Flocks in Egypt.

PubMed

Abdel-Sabour, Mohammed A; Al-Ebshahy, Emad M; Khaliel, Samy A; Abdel-Wanis, Nabil A; Yanai, Tokuma

2017-09-01

The present study aimed to determine the molecular characteristics of circulating infectious bronchitis virus (IBV) strains in vaccinated broiler flocks in the Giza and Fayoum governorates. Thirty-four isolates were collected, and egg propagation revealed their ability to induce typical IBV lesions after three to five successive passages. Three selected isolates were identified as IBV using a real-time reverse transcriptase-PCR assay targeted the nucleocapsid (N) gene and further characterized by partial spike (S) gene sequence analysis. Phylogenetic analysis revealed their clustering into two variant groups. Group I consisted of one variant (VSVRI_F3), which had 99.1% nucleotide sequence identity to the Q1 reference strain. Group II consisted of variants VSVRI_G4 and VSVRI_G9, which showed 92.8%-94.3% nucleotide identity with the Egyptian variants Eg/12120S/2012, Eg/12197B/2012, and Eg/1265B/2012. Regarding the deduced amino acid sequence, the three variants had 77.1%-85.2% similarity with the vaccine strains currently used in Egypt. These findings highlight the importance of monitoring the prevalence of IBV variants in vaccinated broiler flocks as well as adopting an appropriate vaccination strategy.

Prevalence of sand fly fever, West Nile, Crimean-Congo hemorrhagic fever, and leptospirosis antibodies in Pakistani military personnel.

PubMed

Bryan, J P; Iqbal, M; Ksiazek, T G; Ahmed, A; Duncan, J F; Awan, B; Krieg, R E; Riaz, M; Leduc, J W; Nabi, S; Qureshi, M S; Malik, I A; Legters, L J

1996-03-01

To determine the prevalence of antibodies to viral diseases known or suspected to be present in Pakistan, we studied 570 sera from three groups of adults; two of the groups were involved in outbreaks of hepatitis, and the third included men admitted to a hospital for evaluation of febrile illnesses. Immunoglobulin G antileptospiral antibody was found in 1 to 6% of the subjects, with the highest rate in enlisted military personnel hospitalized for febrile illness. One man in the group with febrile illness had significantly elevated immunoglobulin M antileptospiral antibody titers. However, in a group of recruits experiencing suspected non-A, non-B hepatitis, 19 (11%) of 173 had a 4-fold rise in immunoglobulin M antibody to leptospirosis. Antibody to sand fly fever viruses was found in 27 to 70%. Antibody to West Nile virus was present in 33 to 41% of subjects. Antibody reactive with Japanese encephalitis virus was present in 25%, but plaque reduction neutralization tests suggested this to be cross-reaction with West Nile virus. All 212 specimens tested for antibody to Crimean-Congo hemorrhagic fever and Hantaan viruses were negative. This study indicates that diseases known to be prevalent in other areas of southwest Asia and the Middle East are also prevalent in northern Pakistan and may impact on those traveling or working in this area.

Inclusion Bodies Are a Site of Ebolavirus Replication

PubMed Central

Hoenen, Thomas; Shabman, Reed S.; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F.; Becker, Stephan

2012-01-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place. PMID:22915810

Inclusion bodies are a site of ebolavirus replication.

PubMed

Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

2012-11-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Evaluation of the Efficacy, Potential for Vector Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep

PubMed Central

Bennett, Kristine E.; Drolet, Barbara S.; Lindsay, Robbin; Mecham, James O.; Reeves, Will K.; Weingartl, Hana M.; Wilson, William C.

2015-01-01

Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America, there are susceptible ruminant hosts and competent mosquito vectors, yet there are no fully licensed animal vaccines for this arthropod-borne virus, should it be introduced. Studies in sheep and cattle have found the attenuated strain of RVFV, MP-12, to be both safe and efficacious based on early testing, and a 2-year conditional license for use in U.S. livestock has been issued. The purpose of this study was to further determine the vaccine's potential to infect mosquitoes, the duration of humoral immunity to 24 months postvaccination, and the ability to prevent disease and viremia from a virulent challenge. Vaccination experiments conducted in sheep found no evidence of a potential for vector transmission to 4 North American mosquito species. Neutralizing antibodies were elicited, with titers of >1:40 still present at 24 months postvaccination. Vaccinates were protected from clinical signs and detectable viremia after challenge with virulent virus, while control sheep had fever and high-titered viremia extending for 5 days. Antibodies to three viral proteins (nucleocapsid N, the N-terminal half of glycoprotein GN, and the nonstructural protein from the short segment NSs) were also detected to 24 months using competitive enzyme-linked immunosorbent assays. This study demonstrates that the MP-12 vaccine given as a single dose in sheep generates protective immunity to a virulent challenge with antibody duration of at least 2 years, with no evidence of a risk for vector transmission. PMID:26041042

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

PubMed Central

Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2016-01-01

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem–loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR–cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59. PMID:26826129

Insights into the Hendra virus NTAIL-XD complex: Evidence for a parallel organization of the helical MoRE at the XD surface stabilized by a combination of hydrophobic and polar interactions.

PubMed

Erales, Jenny; Beltrandi, Matilde; Roche, Jennifer; Maté, Maria; Longhi, Sonia

2015-08-01

The Hendra virus is a member of the Henipavirus genus within the Paramyxoviridae family. The nucleoprotein, which consists of a structured core and of a C-terminal intrinsically disordered domain (N(TAIL)), encapsidates the viral genome within a helical nucleocapsid. N(TAIL) partly protrudes from the surface of the nucleocapsid being thus capable of interacting with the C-terminal X domain (XD) of the viral phosphoprotein. Interaction with XD implies a molecular recognition element (MoRE) that is located within N(TAIL) residues 470-490, and that undergoes α-helical folding. The MoRE has been proposed to be embedded in the hydrophobic groove delimited by helices α2 and α3 of XD, although experimental data could not discriminate between a parallel and an antiparallel orientation of the MoRE. Previous studies also showed that if the binding interface is enriched in hydrophobic residues, charged residues located close to the interface might play a role in complex formation. Here, we targeted for site directed mutagenesis two acidic and two basic residues within XD and N(TAIL). ITC studies showed that electrostatics plays a crucial role in complex formation and pointed a parallel orientation of the MoRE as more likely. Further support for a parallel orientation was afforded by SAXS studies that made use of two chimeric constructs in which XD and the MoRE were covalently linked to each other. Altogether, these studies unveiled the multiparametric nature of the interactions established within this complex and contribute to shed light onto the molecular features of protein interfaces involving intrinsically disordered regions. Copyright © 2015 Elsevier B.V. All rights reserved.

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

PubMed Central

Etienne, Loïc; Blanchard, Emmanuelle; Boyer, Audrey; Desvignes, Virginie; Gaillard, Julien; Meunier, Jean-Christophe; Roingeard, Philippe; Hourioux, Christophe

2015-01-01

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. PMID:26339783

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

PubMed

Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2016-04-01

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59. © 2016 Belfetmi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines.

PubMed

See, Raymond H; Petric, Martin; Lawrence, David J; Mok, Catherine P Y; Rowe, Thomas; Zitzow, Lois A; Karunakaran, Karuna P; Voss, Thomas G; Brunham, Robert C; Gauldie, Jack; Finlay, B Brett; Roper, Rachel L

2008-09-01

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

PubMed

Widana Gamage, Shirani M K; Dietzgen, Ralf G

2017-01-01

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans -complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D 155 residue in the 30K-like movement protein-specific LxD/N 50-70 G motif of NSm was critical for

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

PubMed

Yang, Qin; Maluf, Nasib Karl; Catalano, Carlos Enrique

2008-11-28

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to

Epidemiology of hantavirus infection in Thousand Islands regency of Jakarta, Indonesia.

PubMed

Ibrahim, Ima-Nurisa; Shimizu, Kenta; Yoshimatsu, Kumiko; Yunianto, Andre; Salwati, Ervi; Yasuda, Shumpei P; Koma, Takaaki; Endo, Rika; Arikawa, Jiro

2013-01-01

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.

Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34.

PubMed

Ott, Melanie; Tascher, Georg; Hassdenteufel, Sarah; Zimmermann, Richard; Haas, Jürgen; Bailer, Susanne M

2011-12-01

Release of herpes simplex virus type 1 (HSV-1) nucleocapsids from the host nucleus relies on the nuclear egress complex consisting of the two essential proteins pUL34 and pUL31. The cytoplasmically exposed N-terminal region of pUL34 interacts with pUL31, while a hydrophobic region followed by a short luminal part mediates membrane association. Based on its domain organization, pUL34 was postulated to be a tail-anchor (TA) protein. We performed a coupled in vitro transcription/translation assay to show that membrane insertion of pUL34 occurs post-translationally. Transient transfection and localization experiments in mammalian cells were combined with HSV-1 bacterial artificial chromosome mutagenesis to reveal the functional properties of the essential pUL34 TA. Our data show that a minimal tail length of 15 residues is sufficient for nuclear envelope targeting and pUL34 function. Permutations of the pUL34 TA with orthologous regions of human cytomegalovirus pUL50 or Epstein-Barr virus pBFRF1 as well as the heterologous HSV-1 TA proteins pUL56 or pUS9 or the cellular TA proteins Bcl-2 and Vamp2 revealed that nuclear egress tolerates TAs varying in sequence and hydrophobicity, while a non-α-helical membrane anchor failed to complement the pUL34 function. In conclusion, this study provides the first mechanistic insights into the particular role of the TA of pUL34 in membrane curving and capsid egress from the host nucleus.

Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

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

Lebrun, Marielle; Thelen, Nicolas; Thiry, Marc

2014-04-15

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond tomore » capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.« less

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

PubMed

Zhu, Tengfei; Song, Hao; Peng, Ruchao; Shi, Yi; Qi, Jianxun; Gao, George F

2017-09-15

Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

PubMed Central

Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

2000-01-01

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection

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

Choi, Yun; Kwon, Young-Chan; Kim, Soo-In

Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by {alpha}v{beta}3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infectionmore » and pathogenesis.« less

Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

PubMed

Mohan, K S; Gopinathan, K P

1989-01-01

Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

PubMed

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

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.

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

PubMed

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

Deletion Mutagenesis Downstream of the 5′ Long Terminal Repeat of Human Immunodeficiency Virus Type 1 Is Compensated for by Point Mutations in both the U5 Region and gag Gene

PubMed Central

Liang, Chen; Rong, Liwei; Russell, Rodney S.; Wainberg, Mark A.

2000-01-01

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5′ long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA3Lys primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels. PMID:10864634

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

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

PubMed

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Hemorrhagic fever with renal syndrome caused by Hantaan virus infection in four pregnant Chinese women.

PubMed

Ji, Fanpu; Zhao, Wenxuan; Liu, Hailing; Zheng, Hongbo; Wang, Shengbang; He, Caini; Wang, Wenjun; Zhang, Ruijuan; Bai, Dan; Tian, Changyin; Zhao, Wenxue; Deng, Hong

2017-10-01

Hantavirus infection during pregnancy can influence both maternal and fetal outcomes. Here, we describe four cases of hemorrhagic fever with renal syndrome (HFRS) in pregnant Chinese women. The HFRS put these women at increased risk for severe illness, despite the patients' symptomologies in the onset phase were similar to those observed in non-pregnant HFRS patients, such as fever, headache, nausea, and thrombocytopenia. Pregnant women appeared to have a more severe status, presenting with severe complications, such as hypervolemia and pulmonary edema. Nevertheless, with appropriate management, mothers with HFRS may carry to full-term and breastfeeding maybe safe and feasible. © 2017 Wiley Periodicals, Inc.

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization.

PubMed

Oglesbee, M; Jackwood, D; Perrine, K; Axthelm, M; Krakowka, S; Rice, J

1986-11-01

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

Insights into the homo-oligomerization properties of N-terminal coiled-coil domain of Ebola virus VP35 protein.

PubMed

Ramaswamy, Venkata Krishnan; Di Palma, Francesco; Vargiu, Attilio V; Corona, Angela; Piano, Dario; Ruggerone, Paolo; Zinzula, Luca; Tramontano, Enzo

2018-03-02

The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

PubMed

Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

2013-05-01

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

Fatal systemic necrotizing infections associated with a novel paramyxovirus, anaconda paramyxovirus, in green anaconda juveniles.

PubMed

Woo, Patrick C Y; Lau, Susanna K P; Martelli, Paolo; Hui, Suk-Wai; Lau, Candy C Y; Fan, Rachel Y Y; Groff, Joseph M; Tam, Emily W T; Chan, Kwok-Hung; Yuen, Kwok-Yung

2014-10-01

Beginning in July 2011, 31 green anaconda (Eunectes murinus) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 × 10(4) to 1.05 × 10(8) copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical "herringbone" appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus, named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3'-N-U-P/V/I-M-F-HN-L-5'. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission. Copyright © 2014, American Society

Fatal Systemic Necrotizing Infections Associated with a Novel Paramyxovirus, Anaconda Paramyxovirus, in Green Anaconda Juveniles

PubMed Central

Lau, Susanna K. P.; Martelli, Paolo; Hui, Suk-Wai; Lau, Candy C. Y.; Fan, Rachel Y. Y.; Groff, Joseph M.; Tam, Emily W. T.; Chan, Kwok-Hung

2014-01-01

Beginning in July 2011, 31 green anaconda (Eunectes murinus) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 × 104 to 1.05 × 108 copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical “herringbone” appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus, named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3′-N-U-P/V/I-M-F-HN-L-5′. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission. PMID:25078906

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

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

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Huang, Yong

2014-03-07

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressedmore » cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.« less

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.

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

You, Jae-Hwan; Reed, Mark L.; Hiscox, Julian A.

The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in themore » C-terminal region of the protein.« less

Recent Evidence of Hantavirus Circulation in the American Tropic

PubMed Central

Montoya-Ruiz, Carolina; Diaz, Francisco J.; Rodas, Juan D.

2014-01-01

Hantaan virus was discovered in Korea during the 1970s while other similar viruses were later reported in Asia and Europe. There was no information about hantavirus human infection in the Americas until 1993 when an outbreak was described in the United States. This event promoted new studies to find hantaviruses in the Americas. At first, many studies were conducted in Brazil, Argentina, Chile, Uruguay and Paraguay, while other Latin American countries began to report the presence of these agents towards the end of the 20th century. More than 30 hantaviruses have been reported in the Western Hemisphere with more frequent cases registered in the southern cone (Argentina, Chile, Uruguay, Paraguay, Bolivia and Brazil). However there was an important outbreak in 2000 in Panama and some rare events have been described in Peru, Venezuela and French Guiana. Since hantaviruses have only recently emerged as a potential threat in the tropical zones of the Americas, this review compiles recent hantavirus reports in Central America, the Caribbean islands and the northern region of South America. These studies have generated the discovery of new hantaviruses and could help to anticipate the presentation of possible future outbreaks in the region. PMID:24638203

Changes in Diversification Patterns and Signatures of Selection during the Evolution of Murinae-Associated Hantaviruses

PubMed Central

Castel, Guillaume; Razzauti, Maria; Jousselin, Emmanuelle; Kergoat, Gael J.; Cosson, Jean-François

2014-01-01

In the last 50 years, hantaviruses have significantly affected public health worldwide, but the exact extent of the distribution of hantavirus diseases, species and lineages and the risk of their emergence into new geographic areas are still poorly known. In particular, the determinants of molecular evolution of hantaviruses circulating in different geographical areas or different host species are poorly documented. Yet, this understanding is essential for the establishment of more accurate scenarios of hantavirus emergence under different climatic and environmental constraints. In this study, we focused on Murinae-associated hantaviruses (mainly Seoul Dobrava and Hantaan virus) using sequences available in GenBank and conducted several complementary phylogenetic inferences. We sought for signatures of selection and changes in patterns and rates of diversification in order to characterize hantaviruses’ molecular evolution at different geographical scales (global and local). We then investigated whether these events were localized in particular geographic areas. Our phylogenetic analyses supported the assumption that RNA virus molecular variations were under strong evolutionary constraints and revealed changes in patterns of diversification during the evolutionary history of hantaviruses. These analyses provide new knowledge on the molecular evolution of hantaviruses at different scales of time and space. PMID:24618811

Recent evidence of hantavirus circulation in the American tropic.

PubMed

Montoya-Ruiz, Carolina; Diaz, Francisco J; Rodas, Juan D

2014-03-14

Hantaan virus was discovered in Korea during the 1970s while other similar viruses were later reported in Asia and Europe. There was no information about hantavirus human infection in the Americas until 1993 when an outbreak was described in the United States. This event promoted new studies to find hantaviruses in the Americas. At first, many studies were conducted in Brazil, Argentina, Chile, Uruguay and Paraguay, while other Latin American countries began to report the presence of these agents towards the end of the 20th century. More than 30 hantaviruses have been reported in the Western Hemisphere with more frequent cases registered in the southern cone (Argentina, Chile, Uruguay, Paraguay, Bolivia and Brazil). However there was an important outbreak in 2000 in Panama and some rare events have been described in Peru, Venezuela and French Guiana. Since hantaviruses have only recently emerged as a potential threat in the tropical zones of the Americas, this review compiles recent hantavirus reports in Central America, the Caribbean islands and the northern region of South America. These studies have generated the discovery of new hantaviruses and could help to anticipate the presentation of possible future outbreaks in the region.

The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging

PubMed Central

Elliott, Richard M.

2014-01-01

Rift Valley fever virus (RVFV, family Bunyaviridae) is a mosquito-borne pathogen of both livestock and humans, found primarily in Sub-Saharan Africa and the Arabian Peninsula. The viral genome comprises two negative-sense (L and M segments) and one ambisense (S segment) RNAs that encode seven proteins. The S segment encodes the nucleocapsid (N) protein in the negative-sense and a nonstructural (NSs) protein in the positive-sense, though NSs cannot be translated directly from the S segment but rather from a specific subgenomic mRNA. Using reverse genetics we generated a virus, designated rMP12:S-Swap, in which the N protein is expressed from the NSs locus and NSs from the N locus within the genomic S RNA. In cells infected with rMP12:S-Swap NSs is expressed at higher levels with respect to N than in cells infected with the parental rMP12 virus. Despite NSs being the main interferon antagonist and determinant of virulence, growth of rMP12:S-Swap was attenuated in mammalian cells and gave a small plaque phenotype. The increased abundance of the NSs protein did not lead to faster inhibition of host cell protein synthesis or host cell transcription in infected mammalian cells. In cultured mosquito cells, however, infection with rMP12:S-Swap resulted in cell death rather than establishment of persistence as seen with rMP12. Finally, altering the composition of the S segment led to a differential packaging ratio of genomic to antigenomic RNA into rMP12:S-Swap virions. Our results highlight the plasticity of the RVFV genome and provide a useful experimental tool to investigate further the packaging mechanism of the segmented genome. PMID:24550727

Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

PubMed Central

Mardassi, H; Athanassious, R; Mounir, S; Dea, S

1994-01-01

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:8143254

Virus-Vectored Influenza Virus Vaccines

PubMed Central

Tripp, Ralph A.; Tompkins, S. Mark

2014-01-01

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus.

PubMed

Mardassi, H; Massie, B; Dea, S

1996-07-01

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised against Escherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line. Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse-chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment. During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures. Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds. The oligomerization process, as analyzed from pulse-chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly