Science.gov

Sample records for arterivirus nonstructural protein

  1. Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses

    SciTech Connect

    Han, Mingyuan; Kim, Chi Yong; Rowland, Raymond R.R.; Fang, Ying; Kim, Daewoo; Yoo, Dongwan

    2014-06-15

    Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV–nsp1 and LDV–nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV–nsp1 remained uncleaved. SHFV–nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV–nsp1αβ and SHFV–nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV–nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV–nsp1β, LDV–nsp1β, EAV–nsp1, and SHFV–nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV–nsp1α, CBP degradation was evident in cells expressing LDV–nsp1α and SHFV–nsp1γ, but no such degradation was observed for EAV–nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ

  2. Structural Biology of the Arterivirus nsp11 Endoribonucleases.

    PubMed

    Zhang, Manfeng; Li, Xiaorong; Deng, Zengqin; Chen, Zhenhang; Liu, Yang; Gao, Yina; Wu, Wei; Chen, Zhongzhou

    2017-01-01

    Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development.

  3. Mapping interactions of Chikungunya virus nonstructural proteins.

    PubMed

    Sreejith, R; Rana, Jyoti; Dudha, Namrata; Kumar, Kapila; Gabrani, Reema; Sharma, Sanjeev K; Gupta, Amita; Vrati, Sudhanshu; Chaudhary, Vijay K; Gupta, Sanjay

    2012-10-01

    The four nonstructural proteins (nsPs1-4) of Chikungunya virus (CHIKV) play important roles involving enzymatic activities and specific interactions with both viral and host components, during different stages of viral pathogenesis. Elucidation of the presence and/or absence of interactions among nsPs in a systematic manner is thus of scientific interest. In the current study, each pair-wise combination among the four nonstructural proteins of CHIKV was systematically analyzed for possible interactions. Six novel protein interactions were identified for CHIKV, using systems such as yeast two-hybrid, GST pull down and ELISA, three of which have not been previously reported for the genus Alphavirus. These interactions form a network of organized associations that suggest the spatial arrangement of nonstructural proteins in the late replicase complex. The study identified novel interactions as well as concurred with previously described associations in related alphaviruses.

  4. The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

    PubMed Central

    van Dinten, Leonie C.; van Tol, Hans; Gorbalenya, Alexander E.; Snijder, Eric J.

    2000-01-01

    Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429→Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429→Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of “hinge spacer,” which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is

  5. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle

    PubMed Central

    Posthuma, Clara C.; Nedialkova, Danny D.; Zevenhoven-Dobbe, Jessika C.; Blokhuis, Jeroen H.; Gorbalenya, Alexander E.; Snijder, Eric J.

    2006-01-01

    The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined. PMID:16439522

  6. Zoonotic Potential of Simian Arteriviruses

    PubMed Central

    Bailey, Adam L.; Lauck, Michael; Sibley, Samuel D.; Friedrich, Thomas C.; Freimer, Nelson B.; Jasinska, Anna J.; Phillips-Conroy, Jane E.; Jolly, Clifford J.; Marx, Preston A.; Apetrei, Cristian; Rogers, Jeffrey

    2015-01-01

    Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be “preemergent” zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur. PMID:26559828

  7. Nonstructural protein 5B of hepatitis C virus.

    PubMed

    Lee, Jong-Ho; Nam, In Young; Myung, Heejoon

    2006-06-30

    Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, a-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.

  8. Alphavirus RNA synthesis and non-structural protein functions.

    PubMed

    Rupp, Jonathan C; Sokoloski, Kevin J; Gebhart, Natasha N; Hardy, Richard W

    2015-09-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

  9. Alphavirus RNA synthesis and non-structural protein functions

    PubMed Central

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field. PMID:26219641

  10. Non-structural proteins of arthropod-borne bunyaviruses: roles and functions.

    PubMed

    Eifan, Saleh; Schnettler, Esther; Dietrich, Isabelle; Kohl, Alain; Blomström, Anne-Lie

    2013-10-04

    Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod.

  11. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  12. Rift Valley fever virus structural and non-structural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Rift Valley fever virus (RVFV) encodes structural proteins, nucleoprotein (N), N-terminus glycoprotein (Gn), C-terminus glycoprotein (Gc) and L protein, 78-kDa and non-structural proteins NSm and NSs. Using the baculovirus system we expressed the full-length coding sequence of N, NSs, NSm, Gc an...

  13. A putative nucleoside triphosphate-binding domain in the nonstructural protein of B19 parvovirus is required for cytotoxicity.

    PubMed Central

    Momoeda, M; Wong, S; Kawase, M; Young, N S; Kajigaya, S

    1994-01-01

    Cytotoxicity secondary to B19 parvovirus infection is due to expression of the viral nonstructural protein. Nonstructural proteins of many parvoviruses contain a well-conserved nucleoside triphosphate (NTP)-binding motif, which has been shown to be essential for a variety of protein functions. We show here that cytotoxicity of the B19 parvovirus nonstructural protein was abolished by single mutations of amino acids within the NTP-binding domain, especially within the A motif, implicating NTP-binding in virus-induced cell death. Images PMID:7966641

  14. A nonstructural protein of feline panleukopenia virus: expression in Escherichia coli and detection of multiple forms in infected cells.

    PubMed Central

    Carlson, J O; Lynde-Maas, M K; Shen, Z D

    1987-01-01

    Sequences coding for the nonstructural protein NS1 of the autonomous parvovirus feline panleukopenia virus were expressed in Escherichia coli as fusion proteins. The fusion proteins were specifically bound by antisera from canine parvovirus-infected dogs. Antisera against one of the fusion proteins bound to several proteins found only in feline panleukopenia virus-infected feline cells. Images PMID:3027392

  15. Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis.

    PubMed

    Murray, Catherine L; Jones, Christopher T; Rice, Charles M

    2008-09-01

    Viruses of the Flaviviridae family, including hepatitis C, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. Recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. We discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. Pinpointing the roles of Flaviviridae proteins in virion production could reveal new avenues for antiviral therapeutics.

  16. [Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

    PubMed

    Kuzmenko, Y V; Starodubova, E S; Karganova, G G; Timofeev, A V; Karpov, V L

    2016-01-01

    The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.

  17. Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1

    PubMed Central

    Shen, Weiran; Deng, Xuefeng; Zou, Wei; Cheng, Fang; Engelhardt, John F.; Yan, Ziying

    2015-01-01

    ABSTRACT Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1′ and D1′, in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1′ and smD1′ at the A1′ and D1′ splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1′ mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1′ mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the

  18. Expression, purification and crystallization of a novel nonstructural protein VP9 from white spot syndrome virus

    SciTech Connect

    Liu, Yang; Sivaraman, J.; Hew, Choy L.

    2006-08-01

    The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. Native protein was purified and crystallized by vapour diffusion. The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. To facilitate purification, a cleavable His{sub 6} tag was introduced at the N-terminus. The native protein was purified and crystallized by vapour diffusion against mother liquor containing 2 M sodium acetate, 100 mM MES pH 6.3, 25 mM cadmium sulfate and 3% glycerol. Crystals were obtained within 7 d and diffracted to 2.2 Å; they belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.13, b = 78.21, c = 78.98 Å and four molecules in the asymmetric unit. The selenomethionine-labelled protein produced isomorphous crystals that diffracted to approximately 3.3 Å.

  19. [Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

    PubMed

    Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

    2015-11-01

    Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

  20. Expression, purification and crystallization of a novel nonstructural protein VP9 from white spot syndrome virus.

    SciTech Connect

    Liu,Y.; Sivaraman, J.; Hew, C.

    2006-01-01

    The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. To facilitate purification, a cleavable His{sub 6} tag was introduced at the N-terminus. The native protein was purified and crystallized by vapor diffusion against mother liquor containing 2 M sodium acetate, 100 mM MES pH 6.3, 25 mM cadmium sulfate and 3% glycerol. Crystals were obtained within 7 d and diffracted to 2.2 Angstroms; they belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.13, b = 78.21, c = 78.98 Angstroms and four molecules in the asymmetric unit. The selenomethionine-labeled protein produced isomorphous crystals that diffracted to approximately 3.3 Angstroms.

  1. Oral Combination Vaccine, Comprising Bifidobacterium Displaying Hepatitis C Virus Nonstructural Protein 3 and Interferon-α, Induces Strong Cellular Immunity Specific to Nonstructural Protein 3 in Mice.

    PubMed

    Kitagawa, Koichi; Omoto, Chika; Oda, Tsugumi; Araki, Ayame; Saito, Hiroki; Shigemura, Katsumi; Katayama, Takane; Hotta, Hak; Shirakawa, Toshiro

    2017-01-23

    We previously generated an oral hepatitis C virus (HCV) vaccine using Bifidobacterium displaying the HCV nonstructural protein 3 (NS3) polypeptide. NS3-specific cellular immunity is important for viral clearance and recovery from HCV infection. In this study, we enhanced the cellular immune responses induced by our oral HCV vaccine, Bifidobacterium longum 2165 (B. longum 2165), by combining interferon-α (IFN-α) as an adjuvant with the vaccine in a mouse experimental model. IFN-α is a widely used cytokine meeting the standard of care (SOC) for HCV infection and plays various immunoregulatory roles. We treated C57BL/6N mice with B. longum 2165 every other day and/or IFN-α twice a week for a month and then analyzed the immune responses using spleen cells. We determined the induction of NS3-specific cellular immunity by cytokine quantification, intracellular cytokine staining, and a cytotoxic T lymphocyte (CTL) assay targeting EL4 tumor cells expressing NS3/4A protein (EL4-NS3/4A). We also treated mice bearing EL4-NS3/4A tumor with the combination therapy in vivo. The results confirmed that the combination therapy of B. longum 2165 and IFN-α induced significantly higher IFN-γ secretion, higher population of CD4(+)T and CD8(+)T cells secreting IFN-γ, and higher CTL activity against EL4-NS3/4A cells compared with the control groups of phosphate-buffered saline, B. longum 2165 alone, and IFN-α alone (p < 0.05). We also confirmed that the combination therapy strongly enhanced tumor growth inhibitory effects in vivo with no serious adverse effects (p < 0.05). These results suggest that the combination of B. longum 2165 and IFN-α could induce a strong cellular immunity specific to NS3 protein as a combination therapy augmenting the current SOC immunotherapy against chronic HCV infection.

  2. Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

    SciTech Connect

    Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee; Afanasiev, Boris; Kittayapong, Pattamaporn; Carlson, Jonathan Corsini, Joe

    2008-04-25

    Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.

  3. Non-Canonical Roles of Dengue Virus Non-Structural Proteins.

    PubMed

    Zeidler, Julianna D; Fernandes-Siqueira, Lorena O; Barbosa, Glauce M; Da Poian, Andrea T

    2017-03-13

    The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV) non-structural proteins (NSPs), which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection.

  4. Non-Canonical Roles of Dengue Virus Non-Structural Proteins

    PubMed Central

    Zeidler, Julianna D.; Fernandes-Siqueira, Lorena O.; Barbosa, Glauce M.; Da Poian, Andrea T.

    2017-01-01

    The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV) non-structural proteins (NSPs), which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection. PMID:28335410

  5. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies.

    PubMed

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin

    2009-01-01

    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.

  6. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However...

  7. A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of non-structural protein 3A in foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10–20 amino acid deletion has been implicated as being responsible for virus attenuation in cattle: a 10 amino acid deletion in t...

  8. Parvovirus B19 Nonstructural Protein-Induced Damage of Cellular DNA and Resultant Apoptosis

    PubMed Central

    Poole, Brian D.; Kivovich, Violetta; Gilbert, Leona; Naides, Stanley J.

    2011-01-01

    Parvovirus B19 is a widespread virus with diverse clinical presentations. The viral nonstructural protein, NS1, binds to and cleaves the viral genome, and induces apoptosis when transfected into nonpermissive cells, such as hepatocytes. We hypothesized that the cytotoxicity of NS1 in such cells results from chromosomal DNA damage caused by the DNA-nicking and DNA-attaching activities of NS1. Upon testing this hypothesis, we found that NS1 covalently binds to cellular DNA and is modified by PARP, an enzyme involved in repairing single-stranded DNA nicks. We furthermore discovered that the DNA nick repair pathway initiated by poly(ADPribose)polymerase and the DNA repair pathways initiated by ATM/ATR are necessary for efficient apoptosis resulting from NS1 expression. PMID:21278893

  9. Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.

    PubMed

    Arima, N; Kao, C Y; Licht, T; Padmanabhan, R; Sasaguri, Y; Padmanabhan, R

    2001-04-20

    Hepatitis C virus nonstructural protein, NS5A, is a phosphoprotein produced from the processing of the viral polyprotein precursor. NS5A associates with several cellular proteins in mammalian cells, and the biological consequences of this interaction are currently unknown. To this end, five stable NS5A-expressing murine and human cell lines were established. Tetracycline-regulated NIH3T3 cells and rat liver epithelial cells as well as the constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3 cells all exhibited cell growth retardation compared with the control cells. Cell cycle analysis by flow cytometry indicated that the NS5A-expressing human epitheloid tumor cells had a reduced S phase and an increase in the G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/Cip1) was found to be in a complex with Cdk2 in the NS5A-expressing human hepatic cell line. Cdk1 and cyclin B1 proteins were also reduced in human Chang liver cells consistent with the increase in G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.

  10. Elicitation of T-cell responses by structural and non-structural proteins of coxsackievirus B4.

    PubMed

    Bengs, Suvi; Marttila, Jane; Susi, Petri; Ilonen, Jorma

    2015-02-01

    Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.

  11. O'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3.

    PubMed

    Saxton-Shaw, Kali D; Ledermann, Jeremy P; Borland, Erin M; Stovall, Janae L; Mossel, Eric C; Singh, Amber J; Wilusz, Jeffrey; Powers, Ann M

    2013-01-01

    O'nyong nyong virus (ONNV) and Chikungunya virus (CHIKV) are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3), was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

  12. Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus.

    PubMed Central

    Christensen, J; Pedersen, M; Aasted, B; Alexandersen, S

    1995-01-01

    We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink. PMID:7853520

  13. A phenotypic assay to identify Chikungunya virus inhibitors targeting the nonstructural protein nsP2.

    PubMed

    Lucas-Hourani, Marianne; Lupan, Alexandru; Desprès, Philippe; Thoret, Sylviane; Pamlard, Olivier; Dubois, Joëlle; Guillou, Catherine; Tangy, Frédéric; Vidalain, Pierre-Olivier; Munier-Lehmann, Hélène

    2013-02-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

  14. Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis.

    PubMed

    Gouklani, Hamed; Bull, Rowena A; Beyer, Claudia; Coulibaly, Fasséli; Gowans, Eric J; Drummer, Heidi E; Netter, Hans J; White, Peter A; Haqshenas, Gholamreza

    2012-05-01

    The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

  15. Hepatitis C Virus Nonstructural Protein 5B Is Involved in Virus Morphogenesis

    PubMed Central

    Gouklani, Hamed; Bull, Rowena A.; Beyer, Claudia; Coulibaly, Fasséli; Gowans, Eric J.; Drummer, Heidi E.; Netter, Hans J.; White, Peter A.

    2012-01-01

    The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis. PMID:22345449

  16. Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

    SciTech Connect

    Khunchai, Sasiprapa; Junking, Mutita; Suttitheptumrong, Aroonroong; Yasamut, Umpa; Sawasdee, Nunghathai; Netsawang, Janjuree; Morchang, Atthapan; Chaowalit, Prapaipit; Noisakran, Sansanee; Yenchitsomanus, Pa-thai; and others

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

  17. Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses

    PubMed Central

    Sironi, Manuela; Forni, Diego; Clerici, Mario; Cagliani, Rachele

    2016-01-01

    The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian

  18. Structure and Function of the Non-Structural Protein of Dengue Virus and its Applications in Antiviral Therapy.

    PubMed

    Xie, Qian; Zhang, Bao; Yu, JianHai; Wu, Qinghua; Yang, Fangji; Cao, Hong; Zhao, Wei

    2017-01-01

    Dengue fever, a type of global and tropical infectious disease, and its prevention has become a challenging issue worldwide. Antibody-dependent enhancement effects and the virus pathogenic mechanism have not yet been fully elucidated, hindering the development of dengue fever prevention and suitable drug treatment. There is currently no specific prevention and therapy in clinical trials, however, in recent years, studies have focused on the pathogenesis and treatment of dengue. Research focusing on dengue virus nonstructural protein in special drugs for the prevention and control of dengue fever is a new progress leading to improved understanding regarding the prevention and control of dengue fever and suitable drugs for the treatment. The main challenges regarding the structure of dengue virus nonstructural protein and the drugs for antiviral therapy are summarized in this paper.

  19. Interaction of foot-and-mouth disease virus non-structural protein 3A with host protein DCTN3 is important for viral virulence in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-structural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular ...

  20. Pharmacoinformatics approach for investigation of alternative potential hepatitis C virus nonstructural protein 5B inhibitors

    PubMed Central

    Mirza, Muhammad Usman; Ghori, Noor-Ul-Huda; Ikram, Nazia; Adil, Abdur Rehman; Manzoor, Sadia

    2015-01-01

    Hepatitis C virus (HCV) is one of the major viruses affecting the world today. It is a highly variable virus, having a rapid reproduction and evolution rate. The variability of genomes is due to hasty replication catalyzed by nonstructural protein 5B (NS5B) which is also a potential target site for the development of anti-HCV agents. Recently, the US Food and Drug Administration approved sofosbuvir as a novel oral NS5B inhibitor for the treatment of HCV. Unfortunately, it is much highlighted for its pricing issues. Hence, there is an urgent need to scrutinize alternate therapies against HCV that are available at affordable price and do not have associated side effects. Such a need is crucial especially in underdeveloped countries. The search for various new bioactive compounds from plants is a key part of pharmaceutical research. In the current study, we applied a pharmacoinformatics-based approach for the identification of active plant-derived compounds against NS5B. The results were compared to docking results of sofosbuvir. The lead compounds with high-binding ligands were further analyzed for pharmacokinetic and pharmacodynamic parameters based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile. The results showed the potential alternative lead compounds that can be developed into commercial drugs having high binding energy and promising ADMET properties. PMID:25848219

  1. Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

    PubMed

    Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

    2016-08-01

    Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection.

  2. Differences in Processing Determinants of Nonstructural Polyprotein and in the Sequence of Nonstructural Protein 3 Affect Neurovirulence of Semliki Forest Virus

    PubMed Central

    Saul, Sirle; Ferguson, Mhairi; Cordonin, Colette; Fragkoudis, Rennos; Ool, Margit; Tamberg, Nele; Sherwood, Karen; Fazakerley, John K.

    2015-01-01

    ABSTRACT The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV. IMPORTANCE SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes

  3. A perspective on targeting non-structural proteins to combat neglected tropical diseases: Dengue, West Nile and Chikungunya viruses.

    PubMed

    Bhakat, Soumendranath; Karubiu, Wilson; Jayaprakash, Venkatesan; Soliman, Mahmoud E S

    2014-11-24

    Neglected tropical diseases are major causes of fatality in poverty stricken regions across Africa, Asia and some part of America. The combined potential health risk associated with arthropod-borne viruses (arboviruses); Dengue virus (DENV), West Nile Virus (WNV) and Chikungunya Virus (CHIKV) is immense. These arboviruses are either emerging or re-emerging in many regions with recent documented outbreaks in the United States. Despite several recent evidences of emergence, currently there are no approved drugs or vaccines available to counter these diseases. Non-structural proteins encoded by these RNA viruses are essential for their replication and maturation and thus may offer ideal targets for developing antiviral drugs. In recent years, several protease inhibitors have been sourced from plant extract, synthesis, computer aided drug design and high throughput screening as well as through drug reposition based approaches to target the non-structural proteins. The protease inhibitors have shown different levels of inhibition and may thus provide template to develop selective and potent drugs against these devastating arboviruses. This review seeks to shed light on the design and development of antiviral drugs against DENV, WNV and CHIKV to date. To the best of our knowledge, this review provides the first comprehensive update on the development of protease inhibitors targeting non-structural proteins of three most devastating arboviruses, DENV, WNV and CHIKV.

  4. Network mapping among the functional domains of Chikungunya virus nonstructural proteins.

    PubMed

    Rana, Jyoti; Rajasekharan, Sreejith; Gulati, Sahil; Dudha, Namrata; Gupta, Amita; Chaudhary, Vijay Kumar; Gupta, Sanjay

    2014-10-01

    Formation of virus specific replicase complex is among the most important steps that determines the fate of viral transcription and replication during Chikungunya virus (CHIKV) infection. In the present study, the authors have computationally generated a 3D structure of CHIKV late replicase complex on the basis of the interactions identified among the domains of CHIKV nonstructural proteins (nsPs) which make up the late replicase complex. The interactions among the domains of CHIKV nsPs were identified using systems such as pull down, protein interaction ELISA, and yeast two-hybrid. The structures of nsPs were generated using I-TASSER and the biological assembly of the replicase complex was determined using ZRANK and RDOCK. A total of 36 interactions among the domains and full length proteins were tested and 12 novel interactions have been identified. These interactions included the homodimerization of nsP1 and nsP4 through their respective C-ter domains; the associations of nsP2 helicase domain and C-ter domain of nsP4 with methyltransferase and membrane binding domains of nsP1; the interaction of nsP2 protease domain with C-ter domain of nsP4; and the interaction of nsP3 macro and alphavirus unique domains with the C-ter domain of nsP1. The novel interactions identified in the current study form a network of organized associations that suggest the spatial arrangement of nsPs in the late replicase complex of CHIKV.

  5. Extensive Positive Selection Drives the Evolution of Nonstructural Proteins in Lineage C Betacoronaviruses

    PubMed Central

    Cagliani, Rachele; Mozzi, Alessandra; Pozzoli, Uberto; Al-Daghri, Nasser; Clerici, Mario; Sironi, Manuela

    2016-01-01

    ABSTRACT Middle East respiratory syndrome-related coronavirus (MERS-CoV) spreads to humans via zoonotic transmission from camels. MERS-CoV belongs to lineage C of betacoronaviruses (betaCoVs), which also includes viruses isolated from bats and hedgehogs. A large portion of the betaCoV genome consists of two open reading frames (ORF1a and ORF1b) that are translated into polyproteins. These are cleaved by viral proteases to generate 16 nonstructural proteins (nsp1 to nsp16) which compose the viral replication-transcription complex. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs. Results indicated widespread positive selection, acting mostly on ORF1a. The proportion of positively selected sites in ORF1a was much higher than that previously reported for the surface-exposed spike protein. Selected sites were unevenly distributed, with nsp3 representing the preferential target. Several pairs of coevolving sites were also detected, possibly indicating epistatic interactions; most of these were located in nsp3. Adaptive evolution at nsp3 is ongoing in MERS-CoV strains, and two selected sites (G720 and R911) were detected in the protease domain. While position 720 is variable in camel-derived viruses, suggesting that the selective event does not represent a specific adaptation to humans, the R911C substitution was observed only in human-derived MERS-CoV isolates, including the viral strain responsible for the recent South Korean outbreak. It will be extremely important to assess whether these changes affect host range or other viral phenotypes. More generally, data herein indicate that CoV nsp3 represents a major selection target and that nsp3 sequencing should be envisaged in monitoring programs and field surveys. IMPORTANCE Both severe acute respiratory syndrome coronavirus (SARS-CoV) and MERS-CoV originated in bats and spread to humans via an intermediate host. This clearly highlights the potential for coronavirus host shifting and the relevance

  6. Genetic Evidence for an Interferon-Antagonistic Function of Rift Valley Fever Virus Nonstructural Protein NSs

    PubMed Central

    Bouloy, Michèle; Janzen, Christian; Vialat, Pierre; Khun, Huot; Pavlovic, Jovan; Huerre, Michel; Haller, Otto

    2001-01-01

    Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR−/− mice lacking the alpha/beta interferon (IFN-α/β) receptor but remained attenuated in IFN-γ receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-α/β production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-α/β and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-α/β production, we infected susceptible IFNAR−/− mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-α/β production. These results demonstrate that the ability of RVFV to inhibit IFN-α/β production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist. PMID:11152510

  7. Antibodies against nonstructural protein 1 protect mice from dengue virus-induced mast cell activation.

    PubMed

    Chu, Ya-Ting; Wan, Shu-Wen; Chang, Yu-Chang; Lee, Chien-Kuo; Wu-Hsieh, Betty A; Anderson, Robert; Lin, Yee-Shin

    2017-02-27

    Dengue virus (DENV) infection causes dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). DHF/DSS patients have been reported to have increased levels of urinary histamine, chymase, and tryptase, which are major granule-associated mediators from mast cells. Previous studies also showed that DENV-infected human mast cells induce production of proinflammatory cytokines and chemokines, suggesting a role played by mast cells in vascular perturbation as well as leukocyte recruitment. In this study, we show that DENV but not UV-inactivated DENV enhanced degranulation of mast cells and production of chemokines (MCP-1, RANTES, and IP-10) in a mouse model. We have previously shown that antibodies (Abs) against a modified DENV nonstructural protein 1 (NS1), designated DJ NS1, provide protection in mice against DENV challenge. In the present study, we investigate the effects of DJ NS1 Abs on mast cell-associated activities. We showed that administration of anti-DJ NS1 Abs into mice resulted in a reduction of mast cell degranulation and macrophage infiltration at local skin DENV infection sites. The production of DENV-induced chemokines (MCP-1, RANTES, and IP-10) and the percentages of tryptase-positive activated mast cells were also reduced by treatment with anti-DJ NS1 Abs. These results indicate that Abs against NS1 protein provide multiple therapeutic benefits, some of which involve modulating DENV-induced mast cell activation.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.10.

  8. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

    PubMed Central

    Tai, C L; Chi, W K; Chen, D S; Hwang, L H

    1996-01-01

    To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far. PMID:8970970

  9. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  10. Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

    PubMed Central

    Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

    2013-01-01

    Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

  11. Kobuviral Non-structural 3A Proteins Act as Molecular Harnesses to Hijack the Host ACBD3 Protein.

    PubMed

    Klima, Martin; Chalupska, Dominika; Różycki, Bartosz; Humpolickova, Jana; Rezabkova, Lenka; Silhan, Jan; Baumlova, Adriana; Dubankova, Anna; Boura, Evzen

    2017-02-07

    Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.

  12. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    PubMed

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

  13. Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

    PubMed Central

    Zhou, Hongli; Wu, Chao; Paranhos-Baccalà, Gláucia; Vernet, Guy; Jin, Qi; Wang, Jianwei; Hung, Tao

    2013-01-01

    Background Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed. Methodology/Principal Findings In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ2 =  44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive. Conclusions/Significance The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis. PMID:23516396

  14. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  15. Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and Autophagy

    PubMed Central

    Chen, Hong-Ru; Chuang, Yung-Chun; Lin, Yee-Shin; Liu, Hsiao-Sheng; Liu, Ching-Chuan; Perng, Guey-Chuen

    2016-01-01

    Dengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS. PMID:27409803

  16. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

    SciTech Connect

    Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

    2007-09-30

    Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

  17. Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization

    PubMed Central

    Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan

    2016-01-01

    ABSTRACT Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To

  18. Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

    PubMed Central

    Hurst-Hess, Kelley R.; Kuo, Lili

    2015-01-01

    ABSTRACT Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCE The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as

  19. Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response.

    PubMed

    Fros, Jelke J; Major, Lee D; Scholte, Florine E M; Gardner, Joy; van Hemert, Martijn J; Suhrbier, Andreas; Pijlman, Gorben P

    2015-03-01

    The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

  20. Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements.

    PubMed

    Gustin, Kortney M; Guan, Bo-Jhih; Dziduszko, Agnieszka; Brian, David A

    2009-06-01

    Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

  1. Identification of potential molecular associations between chikungunya virus non-structural protein 2 and human host proteins.

    PubMed

    Rana, J; Gulati, S; Rajasekharan, S; Gupta, A; Chaudhary, V; Gupta, S

    2017-01-01

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is considered to be the master regulator of viral RNA replication and host responses generated during viral infection. This protein has two main functional domains: an N-terminal domain which exhibits NTPase, RNA triphosphatase and helicase activities and a C-terminal protease domain. Understanding how CHIKV nsP2 interacts with its host proteins is essential for elucidating all the required processes for viral replication and pathogenesis along with the identification of potential targets for antiviral therapy. In current study yeast two-hybrid (Y2H) screening of a human fetal brain cDNA library was performed using nsP2 protein as bait. The analysis identified seven host proteins (CCDC130, CPNE6, POLR2C, MAPK9, EIF4A2, EEF1A1 and EIF3I) as putative interactors of CHIKV nsP2 which were selected for further analysis based on their roles in host cellular machinery. The gene ontology analysis indicates that these proteins are mainly involved in apoptosis, transcription and translational mechanism of host cell. Domain mapping of nsP2 revealed that these associations are not random connections but instead they have functional significance. Further studies to identify the amino acid residues and their chemical interactions that may help in opening new possibilities for preventing these interactions, thus reducing chances of chikungunya infection were performed. This study expands the understanding of CHIKV-host interactions and is important for rational approaches of discovering new antiviral agents.

  2. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    SciTech Connect

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  3. Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

    PubMed Central

    Hu, Xin; Compton, Jaimee R.; Leary, Dagmar H.; Olson, Mark A.; Lee, Michael S.; Cheung, Jonah; Ye, Wenjuan; Ferrer, Mark; Southall, Noel; Jadhav, Ajit; Glass, Pamela J.; Marugan, Juan; Legler, Patricia M.

    2017-01-01

    The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-L-methionine dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, 475NVCWAK480, which differs from papain’s (CGS25CWAFS), and the enzyme lacks a transition state (TS) stabilizing residue homologous to Q19 in papain. To understand the roles of conserved residues in catalysis we determined the structure of the free enzyme, and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme, each conformer may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold reductions in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle Eastern Respiratory virus (MERS). Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat

  4. Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

    PubMed Central

    Scaturro, Pietro; Cortese, Mirko; Chatel-Chaix, Laurent; Fischl, Wolfgang; Bartenschlager, Ralf

    2015-01-01

    Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles. PMID:26562291

  5. Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus.

    PubMed

    Oem, Jae Ku; Kye, Soo Jeong; Lee, Kwang Nyeong; Park, Jong Hyeon; Kim, Yong Joo; Song, Hee Jong; Yeh, Max

    2005-12-01

    It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

  6. Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro

    PubMed Central

    Wu, Yaoxing; Liu, Qingxiang; Zhou, Jie; Xie, Weihong; Chen, Cheng; Wang, Zefang; Yang, Haitao; Cui, Jun

    2017-01-01

    Type I interferon (IFN) serves as the first line of defense against invading pathogens. Inhibition of IFN-triggered signaling cascade by Zika virus (ZIKV) plays a critical role for ZIKV to evade antiviral responses from host cells. Here we demonstrate that ZIKV nonstructural proteins NS1, NS4B and NS2B3 inhibit the induction of IFN and downstream IFN-stimulated genes through diverse strategies. NS1 and NS4B of ZIKV inhibit IFNβ signaling at TANK-binding kinase 1 level, whereas NS2B-NS3 of ZIKV impairs JAK–STAT signaling pathway by degrading Jak1 and reduces virus-induced apoptotic cell death. Furthermore, co-operation of NS1, NS4B and NS2B3 further enhances viral infection by blocking IFN-induced autophagic degradation of NS2B3. Hence, our study reveals a novel antagonistic system employing multiple ZIKV nonstructural proteins in restricting the innate antiviral responses. PMID:28373913

  7. Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro.

    PubMed

    Wu, Yaoxing; Liu, Qingxiang; Zhou, Jie; Xie, Weihong; Chen, Cheng; Wang, Zefang; Yang, Haitao; Cui, Jun

    2017-01-01

    Type I interferon (IFN) serves as the first line of defense against invading pathogens. Inhibition of IFN-triggered signaling cascade by Zika virus (ZIKV) plays a critical role for ZIKV to evade antiviral responses from host cells. Here we demonstrate that ZIKV nonstructural proteins NS1, NS4B and NS2B3 inhibit the induction of IFN and downstream IFN-stimulated genes through diverse strategies. NS1 and NS4B of ZIKV inhibit IFNβ signaling at TANK-binding kinase 1 level, whereas NS2B-NS3 of ZIKV impairs JAK-STAT signaling pathway by degrading Jak1 and reduces virus-induced apoptotic cell death. Furthermore, co-operation of NS1, NS4B and NS2B3 further enhances viral infection by blocking IFN-induced autophagic degradation of NS2B3. Hence, our study reveals a novel antagonistic system employing multiple ZIKV nonstructural proteins in restricting the innate antiviral responses.

  8. Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus.

    PubMed

    Fogeron, Marie-Laure; Paul, David; Jirasko, Vlastimil; Montserret, Roland; Lacabanne, Denis; Molle, Jennifer; Badillo, Aurélie; Boukadida, Célia; Georgeault, Sonia; Roingeard, Philippe; Martin, Annette; Bartenschlager, Ralf; Penin, François; Böckmann, Anja

    2015-12-01

    Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.

  9. Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

  10. 2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

    PubMed Central

    Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

    2015-01-01

    2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

  11. Arteriviruses, Pegiviruses, and Lentiviruses Are Common among Wild African Monkeys

    PubMed Central

    Bailey, Adam L.; Lauck, Michael; Ghai, Ria R.; Nelson, Chase W.; Heimbruch, Katelyn; Hughes, Austin L.; Goldberg, Tony L.; Jasinska, Anna J.; Freimer, Nelson B.; Apetrei, Cristian

    2016-01-01

    ABSTRACT Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts

  12. Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application.

    PubMed

    Chattopadhyay, Soma; Kumar, Abhishek; Mamidi, Prabhudutta; Nayak, Tapas Kumar; Das, Indrani; Chhatai, Jagamohan; Basantray, Itishree; Bramha, Umarani; Maiti, Prasanta Kumar; Singh, Sujay; Suryawanshi, Amol Ratnakar; Chattopadhyay, Subhasis

    2014-04-01

    The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection.

  13. DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model.

    PubMed

    Wada, Takeshi; Kohara, Michinori; Yasutomi, Yasuhiro

    2013-12-05

    Most of the people infected with hepatitis C virus (HCV) develop chronic hepatitis, which in some cases progresses to cirrhosis and ultimately to hepatocellular carcinoma. Although various immunotherapies against the progressive disease status of HCV infection have been studied, a preventive or therapeutic vaccine against this pathogen is still not available. In this study, we constructed a DNA vaccine expressing an HCV structural protein (CN2), non-structural protein (N25) or the empty plasmid DNA as a control and evaluated their efficacy as a candidate HCV vaccine in C57BL/6 and novel genetically modified HCV infection model (HCV-Tg) mice. Strong cellular immune responses to several HCV structural and non-structural proteins, characterized by cytotoxicity and interferon-gamma (IFN-γ) production, were observed in CN2 or N25 DNA vaccine-immunized C57BL/6 mice but not in empty plasmid DNA-administered mice. The therapeutic effects of these DNA vaccines were also examined in HCV-Tg mice that conditionally express HCV proteins in their liver. Though a reduction in cellular immune responses was observed in HCV-Tg mice, there was a significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine but not in mice administered the empty plasmid DNA. Moreover, both CD8(+) and CD4(+) T cells were required for the decrease of HCV protein in the liver. We found that the N25 DNA vaccine improved pathological changes in the liver compared to the empty plasmid DNA. Thus, these DNA vaccines, especially that expressing the non-structural protein gene, may be an alternative approach for treatment of individuals chronically infected with HCV.

  14. Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

    PubMed

    Ivanov, Konstantin A; Ziebuhr, John

    2004-07-01

    The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.

  15. Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3.

    PubMed

    Zhang, Chao; Liu, Xiao-juan; Wu, Kang-cheng; Zheng, Lu-Ping; Ding, Zuo-mei; Li, Fei; Zou, Peng; Yang, Liang; Wu, Jian-guo; Wu, Zu-jian

    2015-11-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana plants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells from Nicotiana benthamiana.

  16. The non-structural protein NS-2 of Bombyx mori parvo-like virus is localized to the nuclear membrane.

    PubMed

    Wang, Fenghua; Hu, Zhaoyang; He, Yuanqing; Li, Guohui; Kong, Jie; Cao, Jian; Chen, Keping; Yao, Qin

    2011-07-01

    Bombyx mori parvo-like virus (BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear. In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-(BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein.

  17. Production of polyclonal antibody to a recombinant non-structural protein Nsp1a of human astrovirus.

    PubMed

    Liu, Chang; Liu, Wen-Hui; Kan, Li-Li; Li, Xin; Li, Yong-gang; Zhao, Wei

    2014-12-01

    Human astrovirus (HAstV) are important pathogens that cause acute viral diarrhea in infants. Little is known about the mechanisms of astrovirus-induced diarrhea. Previous studies have suggested that an apoptosis inducer may be encoded in the non-structural protein (nsP1a) of astrovirus and contribute to virus-induced diarrhea. To study the biological function of nsP1a and to gain further insight into nsP1a protein-host cell interactions, good quality antibodies must be produced. The nsP1agene of HAstV-1 was cloned into a bacterial expression vector Pgex-6P-1. The recombinant plasmid Pgex-6P-nsP1a was transformed into Escherichia coli BL21 (DE3) and expressed as a fusion protein that contains N-terminal GST tags. The expressed recombinant protein was purified and used as an antigen to produce an nsP1a antiserum in rabbits. ELISA was used to detect the titer of specific antibodies. Specificity activity was detected by Western blot and immunofluorescence analysis. The titer of specific antibodies was up to 1:30,000. Western blotting and immunofluorescence analysis indicated that the polyclonal antibody could recognize specifically the HAstV-1 nsP1a protein.

  18. Immune Response of Multiparous Hyper-Immunized Sows against Peptides from Non-Structural and Structural Proteins of PRRSV.

    PubMed

    Rascón-Castelo, Edgar; Burgara-Estrella, Alexel; Reséndiz-Sandoval, Mónica; Hernández-Lugo, Andrés; Hernández, Jesús

    2015-11-27

    The purpose of this study was to evaluate the humoral and cellular responses of commercial multiparous and hyper-immunized sows against peptides from non-structural (nsp) and structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). We selected sows with different numbers of parities from a commercial farm. Management practices on this farm include the use of the MLV commercial vaccine four times per year, plus two vaccinations during the acclimation period. The humoral response was evaluated via the antibody recognition of peptides from nsp and structural proteins, and the cellular response was assessed by measuring the frequency of peptide and PRRSV-specific IFN-gamma-secreting cells (IFNγ-SC). Our results show that sows with six parities have more antibodies against peptides from structural proteins than against peptides from nsp. The analysis of the cellular response revealed that the number of immunizations did not affect the frequency of IFNγ-SC and that the response was stronger against peptides from structural proteins (M protein) than against nsp (nsp2). In summary, these results demonstrate that multiparous, hyper-immunized sows have a stronger immune humoral response to PRRSV structural peptides than nsp, but no differences in IFNγ-SC against the same peptides were observed.

  19. Immune Response of Multiparous Hyper-Immunized Sows against Peptides from Non-Structural and Structural Proteins of PRRSV

    PubMed Central

    Rascón-Castelo, Edgar; Burgara-Estrella, Alexel; Reséndiz-Sandoval, Mónica; Hernández-Lugo, Andrés; Hernández, Jesús

    2015-01-01

    The purpose of this study was to evaluate the humoral and cellular responses of commercial multiparous and hyper-immunized sows against peptides from non-structural (nsp) and structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). We selected sows with different numbers of parities from a commercial farm. Management practices on this farm include the use of the MLV commercial vaccine four times per year, plus two vaccinations during the acclimation period. The humoral response was evaluated via the antibody recognition of peptides from nsp and structural proteins, and the cellular response was assessed by measuring the frequency of peptide and PRRSV-specific IFN-gamma-secreting cells (IFNγ-SC). Our results show that sows with six parities have more antibodies against peptides from structural proteins than against peptides from nsp. The analysis of the cellular response revealed that the number of immunizations did not affect the frequency of IFNγ-SC and that the response was stronger against peptides from structural proteins (M protein) than against nsp (nsp2). In summary, these results demonstrate that multiparous, hyper-immunized sows have a stronger immune humoral response to PRRSV structural peptides than nsp, but no differences in IFNγ-SC against the same peptides were observed. PMID:26633527

  20. Hepatitis C virus non-structural protein-2 activates CXCL-8 transcription through NF-kappaB.

    PubMed

    Oem, J-K; Jackel-Cram, C; Li, Y-P; Kang, H-N; Zhou, Y; Babiuk, L A; Liu, Q

    2008-01-01

    Hepatitis C is a devastating disease worldwide. Proteins encoded by the etiologic agent, hepatitis C virus (HCV), are believed to play important roles in HCV-associated pathogenesis. However, the biological functions of the non-structural protein-2 (NS2) encoded by HCV are not well characterized. Here, we show that HCV NS2 protein activates CXCL-8 (interleukin-8, IL-8) transcription in HepG2 cells as measured by reverse transcription-polymerase chain reaction and IL-8 promoter-luciferase reporter assays. Furthermore, when the kappaB site on the IL-8 promoter was eliminated by mutagenesis or when intracellular NF-kappaB activity was suppressed by an inhibitor, NS2 did not activate the IL-8 promoter, suggesting a role of NF-kappaB in this process. These results prompted us to hypothesize that HCV NS2 might be able to activate NF-kappaB. This hypothesis was tested by determination of NF-kappaB-driven reporter gene expression and NF-kappaB p65 subunit subcellular localization after HCV NS2 expression. Indeed, NS2 could up-regulate NF-kappaB-driven luciferase activity and was associated with p65 nuclear localization. These results demonstrate that HCV NS2 up-regulates IL-8 transcription through NF-kappaB. This newly identified function increases our understanding of the role of HCV NS2 protein in virus-host interactions.

  1. Historical Outbreaks of Simian Hemorrhagic Fever in Captive Macaques Were Caused by Distinct Arteriviruses

    PubMed Central

    Lauck, Michael; Alkhovsky, Sergey V.; Bào, Yīmíng; Bailey, Adam L.; Shevtsova, Zinaida V.; Shchetinin, Alexey M.; Vishnevskaya, Tatyana V.; Lackemeyer, Matthew G.; Postnikova, Elena; Mazur, Steven; Wada, Jiro; Radoshitzky, Sheli R.; Friedrich, Thomas C.; Lapin, Boris A.; Deriabin, Petr G.; Jahrling, Peter B.; Goldberg, Tony L.; O'Connor, David H.

    2015-01-01

    Simian hemorrhagic fever (SHF) is lethal for macaques. Based on clinical presentation and serological diagnosis, all reported SHF outbreaks were thought to be caused by different strains of the same virus, simian hemorrhagic fever virus (SHFV; Arteriviridae). Here we show that the SHF outbreaks in Sukhumi in 1964 and in Alamogordo in 1989 were caused not by SHFV but by two novel divergent arteriviruses. Our results indicate that multiple divergent simian arteriviruses can cause SHF. PMID:25972539

  2. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    PubMed Central

    Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza. PMID:26938453

  3. The Influenza A Virus Non-structural Protein NS1 Upregulates The Expression of Collagen Triple Helix Repeat Containing 1 Protein.

    PubMed

    Zhu, C; Peng, G; Yi, W; Song, H; Liu, F; Liu, X

    2016-12-01

    Influenza A virus (IAV) infection induces a strong immune response and regulates the expression of many host proteins. The collagen triple helix repeat containing 1 (CTHRC1) protein is a secreted protein that exhibits increased expression during the viral infection process. However, the regulatory function of IAV on CTHRC1 expression is obscure. In this study, we investigated the effect of IAV on CTHRC1 expression and its regulatory mechanism. A total of 106 serum specimens from healthy people and 80 serum specimens from patients infected with IAV were collected. The CTHRC1 levels in the sera from the IVA patients and healthy individuals were measured using an enzyme-linked immunosorbent assay (ELISA), and the differences were statistically analysed. A549 cells were infected with the IAV or delNS1 virus. Additionally, A549 cells were cotransfected with a eukaryotic non-structural NS1 protein gene expression plasmid and the CTHRC1 gene promoter reporter plasmid (pCTHRC1-Luc), and, the luciferase activities were assessed. The CTHRC1 mRNA and protein expression were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The serum CTHRC1 level was significantly higher in the IAV patients than in the healthy individuals. IAV upregulated the CTHRC1 mRNA and protein expression. The non-structural NS1 protein specifically activated CTHRC1 gene promoter activity and upregulated CTHRC1 mRNA and protein expression. The activation function had a dose-dependent effect, indicating that influenza virus upregulated CTHRC1 expression through its NS1 protein.

  4. Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

    PubMed Central

    Li, Dan; Lei, Caoqi; Xu, Zhisheng; Yang, Fan; Liu, Huanan; Zhu, Zixiang; Li, Shu; Liu, Xiangtao; Shu, Hongbing; Zheng, Haixue

    2016-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system. PMID:26883855

  5. Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species▿ †

    PubMed Central

    Hristov, Georgi; Krämer, Melanie; Li, Junwei; El-Andaloussi, Nazim; Mora, Rodrigo; Daeffler, Laurent; Zentgraf, Hanswalter; Rommelaere, Jean; Marchini, Antonio

    2010-01-01

    The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity. PMID:20375165

  6. Characterization of Bombyx mori parvo-like virus non-structural protein NS1.

    PubMed

    Li, Guohui; Sun, Chen; Zhang, Junhong; He, Yuanqing; Chen, Huiqing; Kong, Jie; Huang, Guoping; Chen, Keping; Yao, Qin

    2009-12-01

    NS1 gene of Bombyx mori parvo-like virus (China Zhenjiang isolate, BmDNV-Z) codes a predicted 316-amino acid protein, but its function remains unknown. Results of the current study showed that purified recombinant 6 x His-NS1 protein possesses ATP binding, ATPase, DNA binding, and helicase activities. Only one protein was captured in infected Bombyx mori midgut cells against NS1 target protein by employing co-immunoprecipitation, which was identified to be a viral protein by mass spectrometry. The NS1-interacting protein is encoded by BmDNV-Z ORF4 and its molecular is about 100 kD. Analysis of His pull-down confirmed that binding of identified viral protein to purified recombinant 6 x His-NS1 protein in vitro. Taken together, our results indicated that BmDNV-Z NS1 was a multifunctional protein, which may be involved with virus replication.

  7. Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity

    PubMed Central

    Khasnatinov, Maxim A.; Tuplin, Andrew; Gritsun, Dmitri J.; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Gould, Ernest A.

    2016-01-01

    Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras. PMID:27341437

  8. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    PubMed

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  9. A DIVA system based on the detection of antibodies to non-structural protein 3 (NS3) of bluetongue virus.

    PubMed

    Barros, Sílvia C; Cruz, Benedita; Luís, Tiago M; Ramos, Fernanda; Fagulha, Teresa; Duarte, Margarida; Henriques, Margarida; Fevereiro, Miguel

    2009-06-12

    Vaccination programs for the control of bluetongue (BT) in ruminants have limitations due to difficulties in differentiating infected from vaccinated animals (DIVA). To overcome this problem a DIVA test that looks at a differential immune response to bluetongue virus (BTV) non-structural protein 3 (NS3) was developed. The NS3 encoding gene of strain BTV4/22045/PT04 was inserted into expression vector pET-28a and expressed in Escherichia coli strain JM109. Recombinant NS3 protein was used as an antigen in an indirect ELISA (NS3-ELISA) to measure the serologic response to NS3 protein in cattle and sheep. Following a cattle vaccination/challenge experiment with a bivalent inactivated BTV 2-4 vaccine, NS3 antibodies were detected at approximately 15 days post-infection in control unvaccinated animal, while vaccinated animals did not develop detectable NS3 antibodies and, with exception of one, remained negative even after virus challenge. The inactivated vaccine induced antibodies against the major core structural protein (VP7) of BTV as well as neutralizing antibodies in all animals. To evaluate the applicability of NS3-ELISA in field scenario, a total of 562 serum samples collected from uninfected, BTV-infected and vaccinated animals were tested for NS3 antibodies. Taken together, the results confirm that NS3 antibodies were induced to the greatest levels in animals infected with BTV in comparison to the levels induced in animals immunized with inactivated BTV vaccines, implying that antibody response to NS3 allows the differentiation between infected and vaccinated animals.

  10. Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

    PubMed

    Ip, Peng Peng; Boerma, Annemarie; Regts, Joke; Meijerhof, Tjarko; Wilschut, Jan; Nijman, Hans W; Daemen, Toos

    2014-04-01

    An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

  11. Determination of Viremia and Concentration of Circulating Nonstructural Protein 1 in Patients Infected with Dengue Virus in Mexico

    PubMed Central

    de la Cruz-Hernández, Sergio I.; Flores-Aguilar, Hilario; González-Mateos, Silvia; López-Martinez, Irma; Alpuche-Aranda, Celia; Ludert, Juan E.; del Angel, Rosa M.

    2013-01-01

    Higher levels of viremia and circulating nonstructural protein 1 (NS1) have been associated with dengue disease severity. In this study, viremia and circulating NS1 levels were determined in 225 serum samples collected from patients in Mexico infected with dengue virus serotypes 1 and 2 (DENV-1 and DENV-2). Patients with dengue hemorrhagic fever (DHF) who were infected with DENV-1 showed higher levels of circulating NS1 than patients with dengue fever (DF) (P = 0.0175). Moreover, NS1 levels were higher in patients with primary infections with DENV-1 than in patient infected with DENV-2 (P < 0.0001) and in patients with primary infections with DENV-2 than in patients with secondary infections with DENV-2 (P = 0.0051). Unexpectedly, viremia levels were higher in patients with DF than in those with DHF infected with either DENV-1 or DENV-2 (P = 0.0019 and P = 0.001, respectively) and in patients with primary infections than those with secondary DENV-2 infections (P < 0.0001). Results indicate that levels of circulating NS1 vary according to the infecting serotype, immunologic status (primary or secondary infection), and dengue disease severity. PMID:23339203

  12. Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus.

    PubMed

    Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Mir, Carmen; Gebe, John A; Admon, Arie; López, Daniel

    2016-06-01

    Proper antiviral humoral and cellular immune responses require previous recognition of viral antigenic peptides that are bound to HLA class II molecules, which are exposed on the surface of antigen-presenting cells. The helper immune response is critical for the control and the clearance of human respiratory syncytial virus (HRSV) infection, a virus with severe health risk in infected pediatric, immunocompromised, and elderly populations. In this study, using a mass spectrometry analysis of complex HLA class II-bound peptide pools that were isolated from large amounts of HRSV-infected cells, 19 naturally processed HLA-DR ligands, most of them included in a complex nested set of peptides, were identified. Both the immunoprevalence and the immunodominance of the HLA class II response to HRSV were focused on one nonstructural (NS1) and two structural (matrix and mainly fusion) proteins of the infective virus. These findings have clear implications for analysis of the helper immune response as well as for antiviral vaccine design.

  13. Chikungunya virus nonstructural protein 2 inhibits type I/II interferon-stimulated JAK-STAT signaling.

    PubMed

    Fros, Jelke J; Liu, Wen Jun; Prow, Natalie A; Geertsema, Corinne; Ligtenberg, Maarten; Vanlandingham, Dana L; Schnettler, Esther; Vlak, Just M; Suhrbier, Andreas; Khromykh, Alexander A; Pijlman, Gorben P

    2010-10-01

    Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the host's antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.

  14. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    PubMed Central

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  15. A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating

    PubMed Central

    Asare, Emmanuel; Mugavero, JoAnn; Jiang, Ping; Paul, Aniko V.

    2016-01-01

    ABSTRACT The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection. IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these

  16. Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis.

    PubMed

    Zhang, Chengcheng; He, Lei; Kang, Kai; Chen, Heng; Xu, Lei; Zhang, Yanming

    2014-03-01

    Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell's function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

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

    PubMed

    Neuman, Benjamin W

    2016-11-01

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

  18. The nonstructural proteins of Nipah virus play a key role in pathogenicity in experimentally infected animals.

    PubMed

    Yoneda, Misako; Guillaume, Vanessa; Sato, Hiroki; Fujita, Kentaro; Georges-Courbot, Marie-Claude; Ikeda, Fusako; Omi, Mio; Muto-Terao, Yuri; Wild, T Fabian; Kai, Chieko

    2010-09-15

    Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V-), rNiV(C-), and rNiV(W-), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V-) and rNiV(C-) were lower than the other recombinants. The rNiV(V-), rNiV(C-) and rNiV(W-) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V-) and rNiV(C-) but not the rNiV(W-) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.

  19. Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

    PubMed Central

    Kim, Jong-Wook; Hwang, Soon B.

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation. PMID:23861867

  20. Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells

    SciTech Connect

    Garcia-Briones, Mercedes; Rosas, Maria F.; Gonzalez-Magaldi, Monica; Martin-Acebes, Miguel A.; Sobrino, Francisco . E-mail: fsobrino@cbm.uam.es; Armas-Portela, Rosario . E-mail: rarmas@cbm.uam.es

    2006-06-05

    Differences in the kinetics of expression and cell distribution among FMDV non-structural proteins (NSPs) have been observed in BHK-21-infected cells. 3D{sup pol} was the first protein detected by immunofluorescence (1.5 h p.i.), showing a perinuclear distribution. At 2-2.5 h p.i., 2B, 2C, 3B and 3C were detected, mostly exhibiting a punctuated, scattered pattern, while 3A and 3D{sup pol} appeared concentrated at one side of the nucleus. This distribution was exhibited by all the NSPs from 3 h p.i., being 2C and, to a lesser extent, precursors 2BC and 3ABBB, the only proteins detected by Western blotting at that infection time. From 4 h p.i., all mature NSPs as well as precursors 2BC, 3ABBB, 3ABB, 3AB and 3CD{sup pol} were detected by this technique. In spite of their similar immunofluorescence patterns, 2C and 3A co-localized partially by confocal microscopy at 3.5 h p.i., and 3A, but not 2C, co-localized with the ER marker calreticulin, suggesting differences in the distribution of these proteins and/or their precursors as infection proceeded. Transient expression of 2C and 3AB resulted in punctuated fluorescence patterns similar to those found in early infected cells, while 3A showed a more diffuse distribution. A shift towards a fibrous pattern was noticed for 3ABB, while a major change was observed in cells expressing 3ABBB, which displayed a perinuclear fibrous distribution. Interestingly, when co-expressed with 3D{sup pol}, the pattern observed for 3ABBB fluorescence was altered, resembling that exhibited by cells transfected with 3AB. Transient expression of 3D{sup pol} showed a homogeneous cell distribution that included, as determined by confocal microscopy, the nucleus. This was confirmed by the detection of 3D{sup pol} in nuclear fractions of transfected cells. 3D{sup pol} and its precursor 3CD{sup pol} were also detected in nuclear fractions of infected cells, suggesting that these proteins can directly interact with the nucleus during FMDV infection.

  1. Complementation for an essential ancillary nonstructural protein function across parvovirus genera

    PubMed Central

    Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

    2014-01-01

    Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections. PMID:25194919

  2. Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

    PubMed Central

    Randall, Richard E.; Elliott, Richard M.

    2015-01-01

    ABSTRACT Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. IMPORTANCE Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of

  3. Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

    PubMed Central

    Bian, Yixin; Zhao, Shuoxian; Zhu, Shaomei; Zeng, Jinfeng; Li, Tingting; Fu, Yongshui; Wang, Yuanzhan; Zheng, Xin; Zhang, Ling; Wang, Wenjing; Yang, Baocheng; Zhou, Yuanping; Allain, Jean-Pierre; Li, Chengyao

    2013-01-01

    Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection. PMID:23894620

  4. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

    PubMed

    Chuang, Yung-Chun; Lin, Jessica; Lin, Yee-Shin; Wang, Shuying; Yeh, Trai-Ming

    2016-02-01

    Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS.

  5. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    PubMed

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  6. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins

    PubMed Central

    Dhar, Jayeeta; Barik, Sailen

    2016-01-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar. PMID:27905537

  7. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.

    PubMed

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K; Sunil, Sujatha

    2016-11-30

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.

  8. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    PubMed Central

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K.; Sunil, Sujatha

    2016-01-01

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models. PMID:27901124

  9. Structural features of Zika virus non-structural proteins 3 and -5 and its individual domains in solution as well as insights into NS3 inhibition.

    PubMed

    Saw, Wuan Geok; Pan, Ankita; Subramanian Manimekalai, Malathy Sony; Grüber, Gerhard

    2017-02-12

    Zika virus (ZIKV) has emerged as a pathogen of major health concern. The virus relies on its non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA and the nonstructural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. In this study we present for the first time insights into the overall structure of the entire French Polynesia ZIKV NS3 in solution. The protein is elongated and flexible in solution. Solution studies of the individual protease- and helicase domains show the compactness of the two monomeric enzymes as well as the contribution of the 10-residues linker region to the flexibility of the entire NS3. We show also the solution X-ray scattering data of the French Polynesia ZIKV NS5, which is dimeric in solution and switches to oligomers in a concentration-dependent manner. The solution shapes of the MTase and RdRp domains are described. The dimer arrangement of ZIKV NS5 is discussed in terms of its importance for MTase-RdRp communication and concerted interaction with its flexible and monomeric counterpart NS3 during viral replication and capping. The comparison of ZIKV NS3 and -NS5 solution data with the related DENV nonstructural proteins shed light into the similarities and diversities of these classes of enzymes. Finally, the effect of ATPase inhibitors to the enzymatic active ZIKV NS3 and the individual helicase are provided.

  10. Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

    SciTech Connect

    Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.; Bamford, Jaana K.H.; Oksanen, Hanna M.

    2015-08-15

    Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.

  11. Adaptation of yellow fever virus 17D to Vero cells is associated with mutations in structural and non-structural protein genes.

    PubMed

    Beasley, David W C; Morin, Merribeth; Lamb, Ashley R; Hayman, Edward; Watts, Douglas M; Lee, Cynthia K; Trent, Dennis W; Monath, Thomas P

    2013-09-01

    Serial passaging of yellow fever virus 17D in Vero cells was employed to derive seed material for a novel inactivated vaccine, XRX-001. Two independent passaging series identified a novel lysine to arginine mutation at amino acid 160 of the envelope protein, a surface-exposed residue in structural domain I. A third passage series resulted in an isoleucine to methionine mutation at residue 113 of the NS4B protein, a central membrane spanning region of the protein which has previously been associated with Vero cell adaptation of other mosquito-borne flaviviruses. These studies confirm that flavivirus adaptation to growth in Vero cells can be mediated by structural or non-structural protein mutations.

  12. Activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis C virus non-structural protein 2.

    PubMed

    Oem, Jae-Ku; Jackel-Cram, Candice; Li, Yi-Ping; Zhou, Yan; Zhong, Jin; Shimano, Hitoshi; Babiuk, Lorne A; Liu, Qiang

    2008-05-01

    Transcriptional factor sterol regulatory element-binding protein 1c (SREBP-1c) activates the transcription of lipogenic genes, including fatty acid synthase (FAS). Hepatitis C virus (HCV) infection is often associated with lipid accumulation within the liver, known as steatosis in the clinic. The molecular mechanisms of HCV-associated steatosis are not well characterized. Here, we showed that HCV non-structural protein 2 (NS2) activated SREBP-1c transcription in human hepatic Huh-7 cells as measured by using a human SREBP-1c promoter-luciferase reporter plasmid. We further showed that sterol regulatory element (SRE) and liver X receptor element (LXRE) in the SREBP-1c promoter were involved in SREBP-1c activation by HCV NS2. Furthermore, expression of HCV NS2 resulted in the upregulation of FAS transcription. We also showed that FAS upregulation by HCV NS2 was SREBP-1-dependent since deleting the SRE sequence in a FAS promoter and expressing a dominant-negative SREBP-1 abrogated FAS promoter upregulation by HCV NS2. Taken together, our results suggest that HCV NS2 can upregulate the transcription of SREBP-1c and FAS, and thus is probably a contributing factor for HCV-associated steatosis.

  13. Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

    PubMed Central

    Borowski, P; Oehlmann, K; Heiland, M; Laufs, R

    1997-01-01

    Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV. PMID:9060639

  14. Neutralizing Antibodies Protect against Lethal Flavivirus Challenge but Allow for the Development of Active Humoral Immunity to a Nonstructural Virus Protein

    PubMed Central

    Kreil, Thomas R.; Maier, Elisabeth; Fraiss, Sabine; Eibl, Martha M.

    1998-01-01

    Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion. PMID:9525632

  15. Divergent Simian Arteriviruses Cause Simian Hemorrhagic Fever of Differing Severities in Macaques

    PubMed Central

    Moncla, Louise H.; Weiler, Andrea M.; Charlier, Olivia; Rojas, Oscar; Byrum, Russell; Ragland, Dan R.; Cohen, Melanie; Sanford, Hannah B.; Qin, Jing

    2016-01-01

    ABSTRACT Simian hemorrhagic fever (SHF) is a highly lethal disease in captive macaques. Three distinct arteriviruses are known etiological agents of past SHF epizootics, but only one, simian hemorrhagic fever virus (SHFV), has been isolated in cell culture. The natural reservoir(s) of the three viruses have yet to be identified, but African nonhuman primates are suspected. Eleven additional divergent simian arteriviruses have been detected recently in diverse and apparently healthy African cercopithecid monkeys. Here, we report the successful isolation in MARC-145 cell culture of one of these viruses, Kibale red colobus virus 1 (KRCV-1), from serum of a naturally infected red colobus (Procolobus [Piliocolobus] rufomitratus tephrosceles) sampled in Kibale National Park, Uganda. Intramuscular (i.m.) injection of KRCV-1 into four cynomolgus macaques (Macaca fascicularis) resulted in a self-limiting nonlethal disease characterized by depressive behavioral changes, disturbance in coagulation parameters, and liver enzyme elevations. In contrast, i.m. injection of SHFV resulted in typical lethal SHF characterized by mild fever, lethargy, lymphoid depletion, lymphoid and hepatocellular necrosis, low platelet counts, increased liver enzyme concentrations, coagulation abnormalities, and increasing viral loads. As hypothesized based on the genetic and presumed antigenic distance between KRCV-1 and SHFV, all four macaques that had survived KRCV-1 injection died of SHF after subsequent SHFV injection, indicating a lack of protective heterotypic immunity. Our data indicate that SHF is a disease of macaques that in all likelihood can be caused by a number of distinct simian arteriviruses, although with different severity depending on the specific arterivirus involved. Consequently, we recommend that current screening procedures for SHFV in primate-holding facilities be modified to detect all known simian arteriviruses. PMID:26908578

  16. Two Novel Simian Arteriviruses in Captive and Wild Baboons (Papio spp.)

    PubMed Central

    Bailey, Adam L.; Lauck, Michael; Sibley, Samuel D.; Pecotte, Jerilyn; Rice, Karen; Weny, Geoffrey; Tumukunde, Alex; Hyeroba, David; Greene, Justin; Correll, Michael; Gleicher, Michael; Friedrich, Thomas C.; Jahrling, Peter B.; Kuhn, Jens H.; Goldberg, Tony L.; Rogers, Jeffrey

    2014-01-01

    ABSTRACT Since the 1960s, simian hemorrhagic fever virus (SHFV; Nidovirales, Arteriviridae) has caused highly fatal outbreaks of viral hemorrhagic fever in captive Asian macaque colonies. However, the source(s) of these outbreaks and the natural reservoir(s) of this virus remain obscure. Here we report the identification of two novel, highly divergent simian arteriviruses related to SHFV, Mikumi yellow baboon virus 1 (MYBV-1) and Southwest baboon virus 1 (SWBV-1), in wild and captive baboons, respectively, and demonstrate the recent transmission of SWBV-1 among captive baboons. These findings extend our knowledge of the genetic and geographic diversity of the simian arteriviruses, identify baboons as a natural host of these viruses, and provide further evidence that baboons may have played a role in previous outbreaks of simian hemorrhagic fever in macaques, as has long been suspected. This knowledge should aid in the prevention of disease outbreaks in captive macaques and supports the growing body of evidence that suggests that simian arterivirus infections are common in Old World monkeys of many different species throughout Africa. IMPORTANCE Historically, the emergence of primate viruses both in humans and in other primate species has caused devastating outbreaks of disease. One strategy for preventing the emergence of novel primate pathogens is to identify microbes with the potential for cross-species transmission in their natural state within reservoir species from which they might emerge. Here, we detail the discovery and characterization of two related simian members of the Arteriviridae family that have a history of disease emergence and host switching. Our results expand the phylogenetic and geographic range of the simian arteriviruses and define baboons as a natural host for these viruses. Our findings also identify a potential threat to captive macaque colonies by showing that simian arteriviruses are actively circulating in captive baboons. PMID

  17. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  18. Novel β-Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus▿

    PubMed Central

    Almeida, Marcius S.; Johnson, Margaret A.; Herrmann, Torsten; Geralt, Michael; Wüthrich, Kurt

    2007-01-01

    The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel α/β-fold formed by a mixed parallel/antiparallel six-stranded β-barrel, an α-helix covering one opening of the barrel, and a 310-helix alongside the barrel. We further characterized the full-length 179-residue protein and show that the polypeptide segments of residues 1 to 12 and 129 to 179 are flexibly disordered. The structure is analyzed in a search for possible correlations with the recently reported activity of nsp1 in the degradation of mRNA. PMID:17202208

  19. Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

    PubMed Central

    Shirai, M; Akatsuka, T; Pendleton, C D; Houghten, R; Wychowski, C; Mihalik, K; Feinstone, S; Berzofsky, J A

    1992-01-01

    Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the

  20. Cellular microRNA miR-181b inhibits replication of mink enteritis virus by repression of non-structural protein 1 translation.

    PubMed

    Sun, Jia-zeng; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Li, Zhili; Yi, Bao; Mao, Yaping; Hou, Qiang; Liu, Weiquan

    2013-01-01

    Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. Recent studies have showed that microRNAs (miRNAs), small noncoding RNAs of length ranging from 18-23 nucleotides (nt) participate in host-pathogen interaction networks; however, whether or not miRNAs are involved in MEV infection has not been reported. Our study revealed that miRNA miR-181b inhibited replication of MEV in the feline kidney (F81) cell line by targeting the MEV non-structural protein 1 (NS1) messenger RNA (mRNA) coding region, resulting in NS1 translational repression, while MEV infection reduced miR-181b expression. This is the first description of cellular miRNAs modulating MEV infection in F81 cells, providing further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.

  1. Cellular microRNA miR-181b Inhibits Replication of Mink Enteritis Virus by Repression of Non-Structural Protein 1 Translation

    PubMed Central

    Sun, Jia-zeng; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Li, Zhili; Yi, Bao; Mao, Yaping; Hou, Qiang; Liu, Weiquan

    2013-01-01

    Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. Recent studies have showed that microRNAs (miRNAs), small noncoding RNAs of length ranging from 18–23 nucleotides (nt) participate in host-pathogen interaction networks; however, whether or not miRNAs are involved in MEV infection has not been reported. Our study revealed that miRNA miR-181b inhibited replication of MEV in the feline kidney (F81) cell line by targeting the MEV non-structural protein 1 (NS1) messenger RNA (mRNA) coding region, resulting in NS1 translational repression, while MEV infection reduced miR-181b expression. This is the first description of cellular miRNAs modulating MEV infection in F81 cells, providing further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies. PMID:24349084

  2. Marker vaccine potential of foot-and-mouth disease virus with large deletion in the non-structural proteins 3A and 3B.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Misri, Jyoti; Pattnaik, Bramhadev

    2015-11-01

    Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the traditional inactivated vaccines may sometimes contain traces of FMD viral (FMDV) non-structural protein (NSP), therefore, interfering with the NSP-based serological discrimination between infected and vaccinated animals. The availability of marker vaccine for differentiating FMD infected from vaccinated animals (DIVA) would be crucial for the control and subsequent eradication of FMD in India. In this study, we constructed a negative marker FMDV serotype O virus (vaccine strain O IND R2/1975), containing dual deletions of amino acid residues 93-143 and 10-37 in the non-structural proteins 3A and 3B, respectively through reverse genetics approach. The negative marker virus exhibited similar growth kinetics and plaque morphology in cell culture as compared to the wild type virus. In addition, we also developed and evaluated an indirect ELISA (I-ELISA) targeted to the deleted 3AB NSP region (truncated 3AB) which could be used as a companion differential diagnostic assay. The diagnostic sensitivity and specificity of the truncated 3AB I-ELISA were found to be 95.5% and 96%, respectively. The results from this study suggest that the availability negative marker virus and companion diagnostic assay could open a promising new avenue for the application of DIVA compatible marker vaccine for the control of FMD in India.

  3. Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle.

    PubMed

    Paton, David J; de Clercq, Kris; Greiner, Matthias; Dekker, Aldo; Brocchi, Emiliana; Bergmann, Ingrid; Sammin, Donal J; Gubbins, Simon; Parida, Satya

    2006-10-30

    There has been much debate about the use of the so-called "vaccinate-to-live" policy for the control of foot-and-mouth disease (FMD) in Europe, according to which, spread of the FMD virus (FMDV) from future outbreaks could be controlled by a short period of "emergency" vaccination of surrounding herds, reducing the need for large-scale preemptive culling of at-risk animals. Since vaccinated animals may become subclinically infected with FMDV following challenge exposure, it is necessary to either remove all vaccinates (vaccinate-to-kill) or to detect and remove vaccinates in which virus is circulating or has established persistent infections (vaccinate-to-live), in order to rapidly regain the most favoured trading status of FMD-free without vaccination. The latter approach can be supported by testing vaccinated animals for the presence of antibodies to certain non-structural proteins (NSP) of FMDV, which are induced by infection with the virus, but not by vaccination with purified FMD vaccines. Using test sensitivity and specificity data established at a recent workshop on NSP assays [Brocchi E, Bergmann I, Dekker A, Paton DJ, Sammin DJ, Greiner M, et al. Comparative performance of six ELISAs for antibodies to the non-structural proteins of foot-and-mouth disease. Vaccine, in press], this paper examines the ways in which serological testing with NSP ELISAs can be used and interpreted and the effect that this will have on the confidence with which freedom from infection can be demonstrated within guidelines specified by the World Animal Health Organisation and the European Commission.

  4. A multi-species indirect ELISA for detection of non-structural protein 3ABC specific antibodies to foot-and-mouth disease virus.

    PubMed

    Hosamani, M; Basagoudanavar, S H; Tamil Selvan, R P; Das, Varsha; Ngangom, Pravina; Sreenivasa, B P; Hegde, Raveendra; Venkataramanan, R

    2015-04-01

    Reliable diagnostic tests that are able to distinguish infected from vaccinated animals are a critical component of regional control programs for foot-and-mouth disease (FMD) in the affected countries. Non-structural protein (NSP) serology based on the 3ABC protein has been widely used for this purpose, and several kits are commercially available worldwide. This report presents the development of a 3ABC-antigen-based indirect ELISA, employing a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Recombinant 3ABC protein was expressed in insect cells and purified using affinity column chromatography. Using this protein, an indirect ELISA was developed and validated for the detection of NSP antibodies in serum samples collected from animals with different status of FMD. Diagnostic sensitivity and specificity were found to be 95.8 (95 % CI: 92.8-97.8) and 97.45 % (95 % CI: 94.8-99.0), respectively. The in-house ELISA compared well with the commercially available prioCHECK FMDV NS-FMD kit, with a high agreement between the tests, as determined by the kappa coefficient, which was 0.87. The in-house ELISA showed higher sensitivity for detecting vaccinated and subsequently infected animals, when compared to the reference test. Both of the tests were able to detect NSP antibodies as early as 7-8 days after experimental infection.

  5. Identification of a Novel Nonstructural Protein, VP9, from White Spot Syndrome Virus: Its Structure Reveals a Ferredoxin Fold with Specific Metal Binding Sites

    SciTech Connect

    Liu,Y.; Wu, J.; Song, J.; Sivaraman, J.; Hew, C.

    2006-01-01

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP9, a full-length protein of WSSV, encoded by open reading frame wsv230, was identified for the first time in the infected Penaeus monodon shrimp tissues, gill, and stomach as a novel, nonstructural protein by Western blotting, mass spectrometry, and immunoelectron microscopy. Real-time reverse transcription-PCR demonstrated that the transcription of VP9 started from the early to the late stage of WSSV infection as a major mRNA species. The structure of full-length VP9 was determined by both X-ray and nuclear magnetic resonance (NMR) techniques. It is the first structure to be reported for WSSV proteins. The crystal structure of VP9 revealed a ferredoxin fold with divalent metal ion binding sites. Cadmium sulfate was found to be essential for crystallization. The Cd2+ ions were bound between the monomer interfaces of the homodimer. Various divalent metal ions have been titrated against VP9, and their interactions were analyzed using NMR spectroscopy. The titration data indicated that VP9 binds with both Zn2+ and Cd2+. VP9 adopts a similar fold as the DNA binding domain of the papillomavirus E2 protein. Based on our present investigations, we hypothesize that VP9 might be involved in the transcriptional regulation of WSSV, a function similar to that of the E2 protein during papillomavirus infection of the host cells.

  6. Rice ragged stunt virus segment S6-encoded nonstructural protein Pns6 complements cell-to-cell movement of Tobacco mosaic virus-based chimeric virus.

    PubMed

    Wu, Zujian; Wu, Jianguo; Adkins, Scott; Xie, Lianhui; Li, Weimin

    2010-09-01

    The protein(s) that support intercellular movement of Rice ragged stunt virus (RRSV) have not yet been identified. In this study, the role of three nonstructural proteins Pns6, Pns7 and Pns10 in cell-to-cell movement were determined with a movement-deficient Tobacco mosaic virus (TMV) vector. The results showed that only the Pns6 could complement the cell-to-cell movement of the movement-deficient TMV in Nicotiana tabacum Xanthi nc and N. benthamiana plants, and both N- and C-terminal 50 amino acids of Pns6 were essential for the cell-to-cell movement. Transient expression in epidermal cells from N. benthamiana showed that the Pns6-eGFP fusion protein was present predominantly along the cell wall as well as a few punctate sites perhaps indicating plasmodesmata. Taken together with previous finding that the Pns6 has nucleic acid-binding activity (Shao et al., 2004), the possible role of Pns6 in cell-to-cell movement of RRSV were discussed.

  7. The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I.

    PubMed

    Cao, Qian M; Subramaniam, Sakthivel; Ni, Yan-Yan; Cao, Dianjun; Meng, Xiang-Jin

    2016-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important global swine pathogen. Here we demonstrated that PRRSV down-regulates Swine Leukocyte Antigen class I (SLA-I) expression in porcine alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells. To identify the viral protein(s) involved in SLA-I down-regulation, we tested all 22 PRRSV structural and non-structural proteins and identified that Nsp1α and Nsp2TF, and GP3 significantly down-regulated SLA-I expression with Nsp2TF showing the greatest effect. We further generated a panel of mutant viruses in which the Nsp2TF protein synthesis was abolished, and found that the two mutants with disrupted -2 ribosomal frameshifting elements and additional stop codons in the TF domain were unable to down-regulate SLA-I expression. Additionally we demonstrated that the last 68 amino acids of TF domain in Nsp2TF are critical for this function. Collectively, the results indicate a novel function of Nsp2TF in negative modulation of SLA-I expression.

  8. Non-structural protein P6 encoded by rice black-streaked dwarf virus is recruited to viral inclusion bodies by binding to the viroplasm matrix protein P9-1.

    PubMed

    Sun, Liying; Xie, Li; Andika, Ida Bagus; Tan, Zilong; Chen, Jianping

    2013-08-01

    Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when co-expressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

  9. Generation of Mutant Uukuniemi Viruses Lacking the Nonstructural Protein NSs by Reverse Genetics Indicates that NSs Is a Weak Interferon Antagonist

    PubMed Central

    Rezelj, Veronica V.; Överby, Anna K.

    2015-01-01

    ABSTRACT Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools

  10. The effect of bovine rotavirus and its nonstructural protein 4 on ER stress-mediated apoptosis in HeLa and HT-29 cells.

    PubMed

    Goodarzi, Zahra; Soleimanjahi, Hoorieh; Arefian, Ehsan; Saberfar, Esmaeil

    2016-03-01

    Endoplasmic reticulum (ER) plays important roles in multiple cellular processes as well as cell survival and apoptosis. Perturbation of ER functions leads to ER stress and unfolded protein response (UPR). The primary goal of this response is cell survival, but severe ER stress can trigger apoptosis signaling. In tumor cells, chronically activated UPR response provides tumor growth. So, apoptosis induced by the ER stress has been the target for anti-cancer therapy. In this in vitro study, we examined the apoptotic effect associated with ER stress of bovine rotavirus and its nonstructural protein 4 (NSP4) alone in two cancer cell lines. The plasmid pcDNA3.1 encoding NSP4 protein of bovine rotavirus transfected with lipofectamine 2000 into the HeLa and HT-29 cells for protein production. MTT, flow cytometry, and Western blot were used to evaluate the cell viability, apoptosis, and expression level of C/EBP-homologous protein (CHOP) and activated caspase-4. In parallel, the apoptotic effect of the bovine rotavirus associated with ER stress in the infected cells was examined too. The cytotoxic and apoptotic effect of NSP4 protein on the cells were statistically significant compared to the control groups. However, Western blot showed that the expression of the NSP4 protein by recombinant plasmid did not lead to high expression of CHOP and activation of caspase-4. Interestingly, rotavirus not only induced significant apoptosis but also caused an increase in CHOP expression and caspase-4 activation in the infected cells compared to control. As a result, NSP4 protein and bovine rotavirus can be considered a potential novel bio-therapeutic strategy for cancer treatment.

  11. Nonstructural seismic restraint guidelines

    SciTech Connect

    Butler, D.M.; Czapinski, R.H.; Firneno, M.J.; Feemster, H.C.; Fornaciari, N.R.; Hillaire, R.G.; Kinzel, R.L.; Kirk, D.; McMahon, T.T.

    1993-08-01

    The Nonstructural Seismic Restraint Guidelines provide general information about how to secure or restrain items (such as material, equipment, furniture, and tools) in order to prevent injury and property, environmental, or programmatic damage during or following an earthquake. All SNL sites may experience earthquakes of magnitude 6.0 or higher on the Richter scale. Therefore, these guidelines are written for all SNL sites.

  12. 19F NMR Reveals Multiple Conformations at the Dimer Interface of the Non-Structural Protein 1 Effector Domain from Influenza A Virus

    PubMed Central

    Ma, Li-Chung; Swapna, G. V. T.; Leonard, Paul G.; Ladbury, John E.; Krug, Robert M.; Montelione, Gaetano T.

    2014-01-01

    SUMMARY Non-structural protein 1 of influenza A virus, NS1A, is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain (RBD) and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply 19F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan (5-F-Trp), and demonstrate that the 19F signal of Trp187 is a sensitive, direct monitor of the ED helix-helix dimer interface. 19F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein-protein interface, whose rate is over three orders of magnitude faster than the kinetics of ED dimerization. 19F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1Aat concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. PMID:24582435

  13. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    SciTech Connect

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  14. In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions.

    PubMed

    Maroniche, Guillermo A; Mongelli, Vanesa C; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar; del Vas, Mariana

    2012-09-01

    The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  15. Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects.

    PubMed

    Mansuroglu, Z; Josse, T; Gilleron, J; Billecocq, A; Leger, P; Bouloy, M; Bonnefoy, E

    2010-01-01

    Rift Valley fever virus (RVFV) is an emerging, highly pathogenic virus; RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemorrhagic fever in humans, as well as death, abortions, and fetal deformities in animals. RVFV nonstructural NSs protein, a major factor of the virulence, forms filamentous structures in the nuclei of infected cells. In order to further understand RVFV pathology, we investigated, by chromatin immunoprecipitation, immunofluorescence, fluorescence in situ hybridization, and confocal microscopy, the capacity of NSs to interact with the host genome. Our results demonstrate that even though cellular DNA is predominantly excluded from NSs filaments, NSs interacts with some specific DNA regions of the host genome such as clusters of pericentromeric gamma-satellite sequence. Targeting of these sequences by NSs was correlated with the induction of chromosome cohesion and segregation defects in RVFV-infected murine, as well as sheep cells. Using recombinant nonpathogenic virus rZHDeltaNSs210-230, expressing a NSs protein deleted of its region of interaction with cellular factor SAP30, we showed that the NSs-SAP30 interaction was essential for NSs to target pericentromeric sequences, as well as for induction of chromosome segregation defects. The effect of RVFV upon the inheritance of genetic information is discussed with respect to the pathology associated with fetal deformities and abortions, highlighting the main role played by cellular cofactor SAP30 on the establishment of NSs interactions with host DNA sequences and RVFV pathogenesis.

  16. Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-β.

    PubMed

    Bakre, Abhijeet; Wu, Weining; Hiscox, Julian; Spann, Kirsten; Teng, Michael N; Tripp, Ralph A

    2015-11-01

    Human respiratory syncytial virus (RSV) is a major health challenge in the young and elderly owing to the lack of a safe and effective vaccine and proven antiviral drugs. Understanding the mechanisms by which viral genes and proteins modulate the host response to infection is critical for identifying novel disease intervention strategies. In this study, the RSV non-structural protein NS1 was shown to suppress miR-24 expression during infection. Lack of NS1 was linked to increased expression of miR-24, whilst NS1 overexpression suppressed miR-24 expression. NS1 was found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor (TGF)-b pathway to induce cell cycle arrest. Silencing of KLF6 led to increased miR-24 expression via downregulation of TGF-β. Treatment with exogenous TGF-β suppressed miR-24 expression and induced KLF6. Confocal microscopy showed co-localization of KLF6 and RSV NS1. These findings indicated that RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-β, which facilitates RSV replication.

  17. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    SciTech Connect

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  18. Identification of a linear B-cell epitope on non-structural protein 12 of porcine reproductive and respiratory syndrome virus, using a monoclonal antibody.

    PubMed

    Bi, Caihong; Shao, Zengyu; Zhang, Yuanfeng; Hu, Liang; Li, Jiangnan; Huang, Li; Weng, Changjiang

    2017-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. The structure and function of PRRSV nonstructural protein 12 (NSP12) is still unknown. In this study, we produced a monoclonal antibody, named as 1E5, against the NSP12 protein of HP (highly pathogenic) -PRRSV strain HuN4. A series of partially overlapping recombinant NSP12 truncations and synthesized peptides were used to define the epitope recognized by 1E5. We found that (130)KANATSMRFH(139) is the minimal linear epitope and that it is highly conserved among some HP-PRRSV isolates of type 2 PRRSV, but not the classical isolates of type 2 PRRSV or the isolates of type 1 PRRSV. Therefore, 1E5 can be used to establish a valuable tool to distinguish infections with HP-PRRSV isolates of type 2 PRRSV from the classical isolates of type 2 PRRSV and type 1 PRRSV.

  19. Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

    PubMed Central

    Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

    2011-01-01

    Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

  20. Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

    PubMed Central

    Nüesch, Jürg P. F.; Corbau, Romuald; Tattersall, Peter; Rommelaere, Jean

    1998-01-01

    NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C. PMID:9733839

  1. Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity*

    PubMed Central

    Das, Pratyush Kumar; Merits, Andres; Lulla, Aleksei

    2014-01-01

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5′–3′ directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections. PMID:24407286

  2. Development of a Double Antibody Sandwich ELISA for West Nile Virus Detection Using Monoclonal Antibodies against Non-Structural Protein 1

    PubMed Central

    Ding, Xi-Xia; Li, Xiao-Feng; Deng, Yong-Qiang; Guo, Yong-Hui; Hao, Wei; Qin, Cheng-Feng; Fu, Ning

    2014-01-01

    The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans. PMID:25303282

  3. Functional cross-talk between distant domains of chikungunya virus non-structural protein 2 is decisive for its RNA-modulating activity.

    PubMed

    Das, Pratyush Kumar; Merits, Andres; Lulla, Aleksei

    2014-02-28

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5'-3' directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.

  4. Differentiation of West Nile Virus-Infected Animals from Vaccinated Animals by Competitive ELISA Using Monoclonal Antibodies Against Non-Structural Protein 1

    PubMed Central

    Chung, Kyung Min; Song, Jaewhan

    2012-01-01

    Abstract Antibodies against non-structural protein 1 (NS1) are considered to be the most reliable indicator of a present or past infection by West Nile virus (WNV) in animals. In this study, an in-house competitive enzyme-linked immunosorbent assay (NS1-cELISA) utilizing baculovirus-expressed NS1 and monoclonal antibodies against NS1 was established for the detection of antibody responses to NS1 in WNV-infected animals. The assay was validated by the simultaneous detection of early antibody responses to NS1 and the structural envelope protein in animals infected with WNV, or inoculated with inactivated WNV. NS1-cELISA detected WNV antibodies at 6 days post-infection (dpi) in a WNV-infected rabbit (percent inhibition [PI] value of 84.0), and at 10 dpi in a WNV-infected chicken (PI value of 67.0). The NS1-cELISA was able to detect WNV antibodies in sera from all WNV-infected rabbits at 10 dpi (PI value of 79.2±18.0), and from three of four WNV-infected chickens at 14 dpi (PI value of 73.7±22.8). The results of this study demonstrate that the antibody response to NS1 is similar to that against envelope protein in WNV-infected rabbits and chickens, whereas animals inoculated with inactivated WNV develop antibody responses only to the envelope protein but not to NS1. The NS1-cELISA developed here has the potential to be a useful tool for monitoring WNV circulation (i.e., the prevalence of specific antibodies against WNV NS1), by assaying serum samples from regions in which an inactivated vaccine control strategy has been implemented. PMID:22217168

  5. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases.

    PubMed

    Eckei, Laura; Krieg, Sarah; Bütepage, Mareike; Lehmann, Anne; Gross, Annika; Lippok, Barbara; Grimm, Alexander R; Kümmerer, Beate M; Rossetti, Giulia; Lüscher, Bernhard; Verheugd, Patricia

    2017-02-02

    Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

  6. Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

    PubMed

    Pasetto, Anna; Frelin, Lars; Brass, Anette; Yasmeen, Anila; Koh, Sarene; Lohmann, Volker; Bartenschlager, Ralf; Magalhaes, Isabelle; Maeurer, Markus; Sällberg, Matti; Chen, Margaret

    2012-02-01

    Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

  7. X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions*

    PubMed Central

    Chen, Yafang; Savinov, Sergey N.; Mielech, Anna M.; Cao, Thu; Baker, Susan C.; Mesecar, Andrew D.

    2015-01-01

    Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication. PMID:26296883

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

    PubMed

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

    2012-08-01

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

  9. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    PubMed

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  10. The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases

    PubMed Central

    Eckei, Laura; Krieg, Sarah; Bütepage, Mareike; Lehmann, Anne; Gross, Annika; Lippok, Barbara; Grimm, Alexander R.; Kümmerer, Beate M.; Rossetti, Giulia; Lüscher, Bernhard; Verheugd, Patricia

    2017-01-01

    Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict. PMID:28150709

  11. The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner

    PubMed Central

    Marc, Daniel; Barbachou, Sosthène; Soubieux, Denis

    2013-01-01

    Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5′ terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5′-GUAAC / 3′-CUUAG double-stranded motif, which closely resembles the canonical 5′-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar KD values) that leads to oligomerization of the RBD on its RNA ligands. PMID:23093596

  12. Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    PubMed Central

    Okushin, Kazuya; Enooku, Kenichiro; Fujinaga, Hidetaka; Moriya, Kyoji; Yotsuyanagi, Hiroshi; Aizaki, Hideki; Suzuki, Tetsuro; Matsuura, Yoshiharu; Koike, Kazuhiko

    2017-01-01

    The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2’-5’ oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity. PMID:28107512

  13. Nonstructural protein 1{alpha} subunit-based inhibition of NF-{kappa}B activation and suppression of interferon-{beta} production by porcine reproductive and respiratory syndrome virus

    SciTech Connect

    Song Cheng; Krell, Peter; Yoo, Dongwan

    2010-11-25

    Induction of type I interferon (IFN-{alpha}/{beta}) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1{alpha} (Nsp1{alpha}) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-{beta} production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1{alpha} suppressed the activation of nuclear factor (NF)-{kappa}B when stimulated with dsRNA or tumor necrosis factor (TNF)-{alpha}, and NF-{kappa}B suppression was RIG-I-dependent. The suppression of NF-{kappa}B activation was associated with the poor production of IFN-{beta} during PRRSV infection. The C-terminal 14 amino acids of the Nsp1{alpha} subunit were critical in maintaining immunosuppressive activity of Nsp1{alpha} for both IFN-{beta} and NF-{kappa}B, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1{alpha} inhibited I{kappa}B phosphorylation and as a consequence NF-{kappa}B translocation to the nucleus was blocked, leading to the inhibition of NF-{kappa}B stimulated gene expression. Our results suggest that PRRSV Nsp1{alpha} is a multifunctional nuclear protein participating in the modulation of the host IFN system.

  14. Dominant recognition by human CD8+ cytotoxic T lymphocytes of dengue virus nonstructural proteins NS3 and NS1.2a.

    PubMed Central

    Mathew, A; Kurane, I; Rothman, A L; Zeng, L L; Brinton, M A; Ennis, F A

    1996-01-01

    A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8+ T cell responses to dengue viruses. Memory CD8+CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8+CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35.These results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue. PMID:8833919

  15. XAS Characterization of the Zn Site of Non-structural Protein 3 (NS3) from Hepatitis C Virus

    NASA Astrophysics Data System (ADS)

    Ascone, I.; Nobili, G.; Benfatto, M.; Congiu-Castellano, A.

    2007-02-01

    XANES spectra of non structural protein 3 (NS3) have been calculated using 4 Zn coordination models from three crystallographic structures in the Protein Data Base (PDB): 1DY9, subunit B, 1CU1 subunit A and B, and 1JXP subunit B. Results indicate that XANES is an appropriate tool to distinguish among them. Experimental XANES spectra have been simulated refining crystallographic data. The model obtained by XAS is compared with the PDB models.

  16. Diagnostic application of recombinant non-structural protein 3A to detect antibodies induced by foot-and-mouth disease virus infection.

    PubMed

    Biswal, Jitendra K; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-05-01

    Detection of antibodies to the non-structural proteins (NSPs) of FMD virus (FMDV) is the preferred differential diagnostic method for identification of FMD-infected animals in the vaccinated population. Nevertheless, due to the observed variability in the antibody response to NSPs, the likelihood of screening or confirming the FMD infection status in animals is increased if an antibody profile to multiple NSPs is considered for diagnosis. In order to develop and evaluate an additional NSP-based diagnostic assay, in this study, the recombinant 3A protein of FMDV was expressed in Escherichia coli and used as an antigen for detection of FMD infection specific antibodies. At the fixed cut-off value of 45 percentage of positivity, the diagnostic sensitivity and specificity of 3A indirect-ELISA (I-ELISA) were found to be 95.7% and 96.3%, respectively. In FMD naturally infected cattle, about 85% of clinically infected and 75% of asymptomatic in-contact populations were found positive at 13 months post-outbreak. The 3A I-ELISA was further evaluated with the bovine serum samples collected randomly from different parts of the country. Furthermore, the performance of newly developed 3A I-ELISA was compared with the extensively used in-house r3AB3 I-ELISA, and the overall concordance in test results was found to be 93.62%. The r3A I-ELISA could be useful as a screening or confirmatory assay in the sero-surveillance of FMD in India irrespective of extensive bi-annual vaccination.

  17. Systematic analysis of non-structural protein features for the prediction of PTM function potential by artificial neural networks.

    PubMed

    Dewhurst, Henry M; Torres, Matthew P

    2017-01-01

    Post-translational modifications (PTMs) provide an extensible framework for regulation of protein behavior beyond the diversity represented within the genome alone. While the rate of identification of PTMs has rapidly increased in recent years, our knowledge of PTM functionality encompasses less than 5% of this data. We previously developed SAPH-ire (Structural Analysis of PTM Hotspots) for the prioritization of eukaryotic PTMs based on function potential of discrete modified alignment positions (MAPs) in a set of 8 protein families. A proteome-wide expansion of the dataset to all families of PTM-bearing, eukaryotic proteins with a representational crystal structure and the application of artificial neural network (ANN) models demonstrated the broader applicability of this approach. Although structural features of proteins have been repeatedly demonstrated to be predictive of PTM functionality, the availability of adequately resolved 3D structures in the Protein Data Bank (PDB) limits the scope of these methods. In order to bridge this gap and capture the larger set of PTM-bearing proteins without an available, homologous structure, we explored all available MAP features as ANN inputs to identify predictive models that do not rely on 3D protein structural data. This systematic, algorithmic approach explores 8 available input features in exhaustive combinations (247 models; size 2-8). To control for potential bias in random sampling for holdback in training sets, we iterated each model across 100 randomized, sample training and testing sets-yielding 24,700 individual ANNs. The size of the analyzed dataset and iterative generation of ANNs represents the largest and most thorough investigation of predictive models for PTM functionality to date. Comparison of input layer combinations allows us to quantify ANN performance with a high degree of confidence and subsequently select a top-ranked, robust fit model which highlights 3,687 MAPs, including 10,933 PTMs with a high

  18. Systematic analysis of non-structural protein features for the prediction of PTM function potential by artificial neural networks

    PubMed Central

    2017-01-01

    Post-translational modifications (PTMs) provide an extensible framework for regulation of protein behavior beyond the diversity represented within the genome alone. While the rate of identification of PTMs has rapidly increased in recent years, our knowledge of PTM functionality encompasses less than 5% of this data. We previously developed SAPH-ire (Structural Analysis of PTM Hotspots) for the prioritization of eukaryotic PTMs based on function potential of discrete modified alignment positions (MAPs) in a set of 8 protein families. A proteome-wide expansion of the dataset to all families of PTM-bearing, eukaryotic proteins with a representational crystal structure and the application of artificial neural network (ANN) models demonstrated the broader applicability of this approach. Although structural features of proteins have been repeatedly demonstrated to be predictive of PTM functionality, the availability of adequately resolved 3D structures in the Protein Data Bank (PDB) limits the scope of these methods. In order to bridge this gap and capture the larger set of PTM-bearing proteins without an available, homologous structure, we explored all available MAP features as ANN inputs to identify predictive models that do not rely on 3D protein structural data. This systematic, algorithmic approach explores 8 available input features in exhaustive combinations (247 models; size 2–8). To control for potential bias in random sampling for holdback in training sets, we iterated each model across 100 randomized, sample training and testing sets—yielding 24,700 individual ANNs. The size of the analyzed dataset and iterative generation of ANNs represents the largest and most thorough investigation of predictive models for PTM functionality to date. Comparison of input layer combinations allows us to quantify ANN performance with a high degree of confidence and subsequently select a top-ranked, robust fit model which highlights 3,687 MAPs, including 10,933 PTMs with a

  19. Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge.

    PubMed

    Henriques, Hugo R; Rampazo, Eline V; Gonçalves, Antonio J S; Vicentin, Elaine C M; Amorim, Jaime H; Panatieri, Raquel H; Amorim, Kelly N S; Yamamoto, Marcio M; Ferreira, Luís C S; Alves, Ada M B; Boscardin, Silvia B

    2013-01-01

    Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens

  20. The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

    PubMed Central

    Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling

    2015-01-01

    ABSTRACT NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. IMPORTANCE The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice

  1. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    SciTech Connect

    Sun, Bin; Cai, Yingyue; Li, Yongshu; Li, Jingjing; Liu, Kaiyu; Li, Yi; Yang, Yongbo

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  2. Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody.

    PubMed

    Wu, Jianping; Mok, Chee-Keng; Chow, Vincent Tak Kwong; Yuan, Y Adam; Tan, Yee-Joo

    2016-09-16

    We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.

  3. Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody

    PubMed Central

    Wu, Jianping; Mok, Chee-Keng; Chow, Vincent Tak Kwong; Yuan, Y. Adam; Tan, Yee-Joo

    2016-01-01

    We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA. PMID:27633136

  4. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    PubMed

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules.

  5. Increased phosphoenolpyruvate carboxykinase gene expression and steatosis during hepatitis C virus subgenome replication: role of nonstructural component 5A and CCAAT/enhancer-binding protein β.

    PubMed

    Qadri, Ishtiaq; Choudhury, Mahua; Rahman, Shaikh Mizanoor; Knotts, Trina A; Janssen, Rachel C; Schaack, Jerome; Iwahashi, Mieko; Puljak, Livia; Simon, Francis R; Kilic, Gordan; Fitz, J Gregory; Friedman, Jacob E

    2012-10-26

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression

  6. Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses

    PubMed Central

    Whelan, Jillian N.; Tran, Kim C.; van Rossum, Damian B.

    2016-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. IMPORTANCE RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance

  7. Evaluation of Non-Structural Protein-1(NS1) positive patients of 2013 dengue outbreak in Khyber Pakhtunkhwa, Pakistan

    PubMed Central

    Lutfullah, Ghosia; Ahmed, Jawad; Khan, Aftab; Ihsan, Hina; Ahmad, Jamshed

    2017-01-01

    Background & Objective: Dengue infection is an arthropod borne disease caused by Dengue virus in humans. Dengue virus infection has more potential to produce severe form of the disease with more severe symptoms. Proper diagnosis of dengue fever is very important for its safe management. The objective of this study was to evaluate the non structural protein-1 (NS1) positive parameter for identification of dengue fever by using ELISA from 2013 dengue outbreak in Khyber Pakhtunkhwa. Methods: It was a cross sectional study conducted among 384 patients tested for dengue admitted to different hospitals of Khyber Pakhtunkhwa April to December 2013 with symptoms related to classical dengue fever. Written informed consent was taken from 100 NS1 positive diagnosed patients, and 3 to 5 ml blood sample was collected for confirmation through ELISA testing. ELISA test for dengue IgG and IgM was performed two time in order to confirm the dengue cases. Data was entered and analyzed by using SPSS version 16. Result: The study performed on 100 NS1 positive samples of patients, admitted to hospitals with symptoms related to classical dengue fever, indicated that after performing the IgM and IgG capture ELISA test only 76 samples were actually found positive for dengue. The rest of the 24 samples were found negative for both IgM and IgG capture ELISAs. The study also revealed that 90.8 % patients had primary dengue infection and 35.5% patients had secondary dengue infection. Most patients were between the age of 10-20 years (26%), among them19.7% were having primary dengue infection. Among 10-20 years of age 50% female patients were false dengue patients. Conclusion: About 24 % NSI protein positive samples were found negative for both IgM and IgG capture ELISAs showed that NS1protein positivity does not confirm actual dengue infection. PMID:28367194

  8. Novel PKCη Is Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

    PubMed Central

    Lachmann, Sylvie; Rommeleare, Jean; Nüesch, Jürg P. F.

    2003-01-01

    The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCη in the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle. PMID:12829844

  9. NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

    PubMed

    Melekis, Efstathios; Tsika, Aikaterini C; Lichière, Julie; Chasapis, Christos T; Margiolaki, Irene; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-04-01

    Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

  10. Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins.

    PubMed

    James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W

    2013-12-01

    Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

  11. Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses

    PubMed Central

    Lehmann, Kathleen C.; Gulyaeva, Anastasia; Zevenhoven-Dobbe, Jessika C.; Janssen, George M. C.; Ruben, Mark; Overkleeft, Hermen S.; van Veelen, Peter A.; Samborskiy, Dmitry V.; Kravchenko, Alexander A.; Leontovich, Andrey M.; Sidorov, Igor A.; Snijder, Eric J.; Posthuma, Clara C.; Gorbalenya, Alexander E.

    2015-01-01

    RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies. PMID:26304538

  12. Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

    PubMed Central

    Xie, Zhengyuan; Xiao, Zhihua; Wang, Fenfen

    2017-01-01

    The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor (TNF)-α. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of NF-κB and miR-503. We found that overexpression of NS5A inhibited TNF-αinduced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the TNF-α induced Hep-mock cells was significantly less than the viability of the TNF-α induced Hep-NS5A cells, which demonstrates that NS5A inhibited TNF-α-induced HepG2 cell apoptosis. Under TNF-α treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited TNF-α-induced NF-κB activation and NF-κB regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse TNF-α-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits NF-κB activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis. PMID:28343379

  13. Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA Vaccine.

    PubMed

    Bao, Huihui; Ramanathan, Aarti A; Kawalakar, Omkar; Sundaram, Senthil G; Tingey, Colleen; Bian, Charoran B; Muruganandam, Nagarajan; Vijayachari, Paluru; Sardesai, Niranjan Y; Weiner, David B; Ugen, Kenneth E; Muthumani, Karuppiah

    2013-02-01

    Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

  14. Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent.

    PubMed

    Mohanty, Nihar Nalini; Chacko, Nirmal; Biswas, Sanchay Kumar; Chand, Karam; Pandey, Awadh Bihari; Mondal, Bimalendu; Hemadri, Divakar; Shivachandra, Sathish Bhadravati

    2016-09-01

    Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.

  15. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    SciTech Connect

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-07-05

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  16. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response.

    PubMed

    Moraes, Mauro Pires; Segundo, Fayna Diaz-San; Dias, Camila C; Pena, Lindomar; Grubman, Marvin J

    2011-11-28

    We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference

  17. Functional analysis of the porcine USP18 and its role during porcine arterivirus replication.

    PubMed

    Ait-Ali, Tahar; Wilson, Alison W; Finlayson, Heather; Carré, Wilfrid; Ramaiahgari, Sreenivasa Chakravarthy; Westcott, David G; Waterfall, Martin; Frossard, Jean-Pierre; Baek, Kwang-Hyun; Drew, Trevor W; Bishop, Stephen C; Archibald, Alan L

    2009-06-15

    Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV). We have shown that UBP gene is the ortholog of the murine USP18 (Ubp43) gene and the human ubiquitin specific protease 18 (USP18) gene and encodes a biochemically functional de-ubiquitin enzyme which inhibits signalling pathways that lead to IFN-stimulating response element (ISRE) promotor regulation. Furthermore we have demonstrated that overexpression of the porcine USP18 leads to reduced replication and/or growth of PRRSV. Our data contrast with the conclusion of numerous reports demonstrating that USP18-deficient mice are highly resistant to viral and bacterial infections and to oncogenic transformation by BCR-ABL, and highlight USP18 as a potential target gene that promotes reduced replication of PRRSV.

  18. Porcine Reproductive and Respiratory Syndrome Virus Replicase - Isoforms of Nonstructural Protein 2 and Interaction with Heat Shock 70kDa Protein 5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV), when expressed independently, was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G**1196|G**1197 dipeptide in transfected CHO cells. The proteolytic cleavage of nsp...

  19. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine

    PubMed Central

    Fu, Yuanfang; Li, Pinghua; Cao, Yimei; Wang, Na; Sun, Pu; Shi, Qian; Ji, Xincheng; Bao, Huifang; Li, Dong; Chen, Yingli; Bai, Xingwen; Ma, Xueqing; Zhang, Jing; Lu, Zengjun; Liu, Zaixin

    2017-01-01

    Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved “AEKNPLE” epitope spanning amino acids 109–115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant “AEKNPLE” epitope in NSP 3A of FMDV. PMID:28107470

  20. Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

    PubMed

    Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

    2013-12-01

    Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV.

  1. Electrically tunable absorber based on nonstructured graphene

    NASA Astrophysics Data System (ADS)

    Ye, Caiyan; Zhu, Zhihong; Xu, Wei; Yuan, Xiaodong; Qin, Shiqiao

    2015-12-01

    We demonstrate numerically that a tunable absorber with absorption of 99.94% in the far infrared range can be obtained using a nonstructured graphene. The mechanism originates from a nonstructured graphene film supported on a periodical dielectric array that can show Fermi level modulation periodically and produce plasmonic resonances in the far infrared range. The nonstructured graphene can avoid the unexpected edge effects and does not influence the unique properties of graphene, which will be helpful in practice to achieve the unity absorption and facilitate the development of many related applications.

  2. Production of antiserum to a non-structural potyviral protein and its use to detect narcissus yellow stripe and other potyviruses.

    PubMed

    Mowat, W P; Dawson, S; Duncan, G H

    1989-08-01

    A protein, of apparent molecular weight 72,000, was purified from experimentally infected narcissus plants with yellow stripe symptoms utilising SDS-polyacrylamide gel electrophoresis. This protein was excised from the gels and used to prepare antiserum, which reacted specifically with cytoplasmic cylindrical inclusions in ultra-thin sections of virus-infected cells and, in immunoblots, with the 72 kDa protein in preparations containing cytoplasmic inclusions. The antiserum reacted in ELISA with leaf extracts from yellow stripe diseased plants of four narcissus cultivars but not with extracts from comparable symptomless plants. In tests with extracts of plants infected with seven definitive potyviruses, reactions were obtained with bean yellow mosaic and iris mild mosaic viruses. Virus-specific reactions in dot-blot ELISA were dependent on the presence of Tween 20 in the extraction buffer. In contrast, an antiserum to the putative cytoplasmic inclusion protein of alstroemeria mosaic virus reacted only with SDS-treated leaf extracts of infected plants. In limited tests, the method of purifying cytoplasmic inclusion protein was successfully applied to four definitive potyviruses, suggesting that it may be generally applicable to potyviruses and of use for preparing antisera when purification of virus particles is difficult.

  3. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    NASA Astrophysics Data System (ADS)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  4. A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly.

    PubMed

    Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

    2011-07-01

    Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.

  5. Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses.

    PubMed

    Yin, Cuifeng; Khan, Javed A; Swapna, G V T; Ertekin, Asli; Krug, Robert M; Tong, Liang; Montelione, Gaetano T

    2007-07-13

    Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-A x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier "working models" of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an approximately 45 degrees angle relative to the axes of helices alpha2/alpha2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

  6. Conserved Surface Features Form the Double-stranded RNA Binding Site of Non-structural Protein 1 (NS1) from Influenza A and B Viruses

    SciTech Connect

    Yin,C.; Khan, J.; Swapna, G.; Ertekin, A.; Krug, R.; Tong, L.; Montelione, G.

    2007-01-01

    Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-{angstrom} x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier 'working models' of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an {approx}45{sup o} angle relative to the axes of helices {alpha}2/{alpha}2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

  7. Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

    SciTech Connect

    Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

    2010-03-01

    The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1alpha and nsp1beta subunits. In infected cells, we detected the actual existence of nsp1alpha and nsp1beta. Cleavage sites between nsp1alpha/nsp1beta and nsp1beta/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1alpha and nsp1beta mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-beta expression. The nsp1beta was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1beta has ability to inhibit both interferon synthesis and signaling, while nsp1alpha alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

  8. Equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein 1 as a main interferon antagonist.

    PubMed

    Go, Yun Young; Li, Yanhua; Chen, Zhenhai; Han, Mingyuan; Yoo, Dongwan; Fang, Ying; Balasuriya, Udeni B R

    2014-01-01

    The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.

  9. Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

    PubMed Central

    Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

    2015-01-01

    ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome

  10. School Facilities Manual: Nonstructural Protection Guide. Safer Schools, Earthquake Hazards, Nonstructural. Second Edition.

    ERIC Educational Resources Information Center

    Noson, Linda Lawrance; Perbix, Todd W.

    This guide addresses the strengthening of nonstructural elements of a school building to resist earthquake-induced damaged and improve school building safety in Washington State regions with notable earthquake activity. Nonstructural elements include the decorative details and those functional building parts and contents which support the…

  11. Role of amphotericin B upon enhancement of protective immunity elicited by oral administration with liposome-encapsulated-Japanese encephalitis virus nonstructural protein 1 (NS1) in mice.

    PubMed

    Lin, Tsung-Shun; Chuang, Chuan-Chang; Hsu, Hui-Ling; Liu, Yu-Tien; Lin, Wen-Po; Liang, Chung-Chih; Liu, Wen-Tssann

    2010-09-01

    Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 microg ml(-1)). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 microg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-kappaB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer's-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1beta, IL-6 and TNF-alpha expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to "fine-tune" humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.

  12. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus.

    PubMed

    Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

    2017-02-18

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126-LQxxLxxxGL-135. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export.

  13. Limited effect on NS3-NS4A protein cleavage after alanine substitutions within the immunodominant HLA-A2-restricted epitope of the hepatitis C virus genotype 3a non-structural 3/4A protease.

    PubMed

    Ahlén, Gustaf; Chen, Antony; Roe, Barbara; Falkeborn, Tina; Frelin, Lars; Hall, William W; Sällberg, Matti; Söderholm, Jonas

    2012-08-01

    It has been well established that immunological escape mutations within the hepatitis C virus genotype (gt) 1a non-structural (NS) 3/4A protease are partly prevented by a reduction in viral protease fitness. Surprisingly little is known about whether similar mutations affect proteases from other genotypes. In the present study, we assessed both the HLA-A2-restricted CTL response and gt3a NS3/4A protease fitness. Similar to gt1, the 1073-1081 epitope was immunodominant within the gt3a-specific HLA-A2-restricted CTL response, despite sequence similarity of only 56 % between the gt1a and gt3a genes. However, unlike the gt1a NS3/4A protease, all residues within the gt3a 1073-1081 epitope could be replaced sequentially by alanine while retaining protease activity, at least in part.

  14. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    SciTech Connect

    Lee, Sang-Myeong; Kleiboeker, Steven B. . E-mail: KleiboekerS@Missouri.edu

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradation of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  15. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  16. Nonstructural carbohydrates and return bloom potential differ among cranberry cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    explain low fruit set and biennial bearing tendencies of cranberry (Vaccinium macrocarpon). Yet, comparisons of nonstructural carbohydrate concentrations during critical phenological stages across cultivars that differ in biennial bearing tendencies and return bloom potential are lacking, particular...

  17. Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production

    PubMed Central

    Sun, Yan; Ke, Hanzhong; Han, Mingyuan; Chen, Ning; Fang, Weihuan; Yoo, Dongwan

    2016-01-01

    Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production. PMID:27997564

  18. NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

    PubMed

    Makrynitsa, Garyfallia I; Ntonti, Dioni; Marousis, Konstantinos D; Tsika, Aikaterini C; Lichière, Julie; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-10-01

    Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

  19. Guide and Checklist for Nonstructural Earthquake Hazards in California Schools.

    ERIC Educational Resources Information Center

    2003

    The recommendations included in this document are intended to reduce seismic hazards associated with the non-structural components of schools buildings, including mechanical systems, ceiling systems, partitions, light fixtures, furnishings, and other building contents. It identifies potential earthquake hazards and provides recommendations for…

  20. Reducing Nonstructural Earthquake Damage: A Practical Guide for Schools. [Videotape].

    ERIC Educational Resources Information Center

    Federal Emergency Management Agency, Washington, DC.

    This videotape describes the nonstructural areas within a school that can be damaged and create hazards for students, teachers, and staff during and after an earthquake; and discusses preventive measures to lower the injury potential from these hazards. It confirms that the best procedure to use during an earthquake to protect oneself from…

  1. Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

    PubMed Central

    Aleshin, SE; Timofeev, AV; Khoretonenko, MV; Zakharova, LG; Pashvykina, GV; Stephenson, JR; Shneider, AM; Altstein, AD

    2005-01-01

    Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines. PMID:16076390

  2. Some Aspects of Multigrid Methods on Non-Structured Meshes

    NASA Technical Reports Server (NTRS)

    Guillard, H.; Marco, N.

    1996-01-01

    To solve a given fine mesh problem, the design of a multigrid method requires the definition of coarse levels, associated coarse grid operators and inter-grid transfer operators. For non-structured simplified meshes, these definitions can rely on the use of non-nested triangulations. These definitions can also be founded on agglomeration/aggregation techniques in a purely algebraic manner. This paper analyzes these two options, shows the connections of the volume-agglomeration method with algebraic methods and proposes a new definition of prolongation operator suitable for the application of the volume-agglomeration method to elliptic problems.

  3. Mentoring an Undergraduate Research Student in the Structural and Nonstructural Properties of Drugs

    ERIC Educational Resources Information Center

    Ealy, Julie B.; Kvarta, Veronica

    2006-01-01

    An undergraduate research student was given the opportunity to take of ownership of her scientific knowledge by helping her to conduct research in the structural and nonstructural properties of drugs. The research highlighted that ketoprofen can be used to illustrate the structural and nonstructural properties of the research, helping the student…

  4. Reducing the Risks of Nonstructural Earthquake Damage: A Practical Guide. Earthquake Hazards Reduction Series 1.

    ERIC Educational Resources Information Center

    Reitherman, Robert

    The purpose of this booklet is to provide practical information to owners, operators, and occupants of office and commercial buildings on the vulnerabilities posed by earthquake damage to nonstructural items and the means available to deal with these potential problems. Examples of dangerous nonstructural damages that have occurred in past…

  5. The KnowRISK project: Tools and strategies to reduce non-structural damage

    NASA Astrophysics Data System (ADS)

    Sousa Oliveira, Carlos; Lopes, Mário; Mota de Sá, Francisco; Amaral Ferreia, Mónica; Candeias, Paulo; Campos Costa, Alfredo; Rupakhety, Rajesh; Meroni, Fabrizio; Azzaro, Raffaele; D'Amico, Salvatore; Langer, Horst; Musacchio, Gemma; Sousa Silva, Delta; Falsaperla, Susanna; Scarfì, Luciano; Tusa, Giuseppina; Tuvé, Tiziana

    2016-04-01

    The project KnowRISK (Know your city, Reduce seISmic risK through non-structural elements) is financed by the European Commission to develop prevention measures that may reduce non-structural damage in urban areas. Pilot areas of the project are within the three European participating countries, namely Portugal, Iceland and Italy. Non-structural components of a building include all those components that are not part of the structural system, more specifically the architectural, mechanical, electrical, and plumbing systems, as well as furniture, fixtures, equipment, and contents. Windows, partitions, granite veneer, piping, ceilings, air conditioning ducts and equipment, elevators, computer and hospital equipment, file cabinets, and retail merchandise are all examples of non-structural components that are vulnerable to earthquake damage. We will use the experience gained during past earthquakes, which struck in particular Iceland, Italy and Portugal (Azores). Securing the non-structural elements improves the safety during an earthquake and saves lives. This paper aims at identifying non-structural seismic protection measures in the pilot areas and to develop a portfolio of good practices for the most common and serious non-structural vulnerabilities. This systematic identification and the portfolio will be achieved through a "cross-knowledge" strategy based on previous researches, evidence of non-structural damage in past earthquakes. Shake table tests of a group of non-structural elements will be performed. These tests will be filmed and, jointly with portfolio, will serve as didactic supporting tools to be used in workshops with building construction stakeholders and in risk communication activities. A Practical Guide for non-structural risk reduction will be specifically prepared for citizens on the basis of the outputs of the project, taking into account the local culture and needs of each participating country.

  6. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis.

    PubMed Central

    Gorbalenya, A E; Koonin, E V; Donchenko, A P; Blinov, V M

    1989-01-01

    Amino acid sequences of 2 giant non-structural polyproteins (F1 and F2) of infectious bronchitis virus (IBV), a member of Coronaviridae, were compared, by computer-assisted methods, to sequences of a number of other positive strand RNA viral and cellular proteins. By this approach, juxtaposed putative RNA-dependent RNA polymerase, nucleic acid binding ("finger"-like) and RNA helicase domains were identified in F2. Together, these domains might constitute the core of the protein complex involved in the primer-dependent transcription, replication and recombination of coronaviruses. In F1, two cysteine protease-like domains and a growth factor-like one were revealed. One of the putative proteases of IBV is similar to 3C proteases of picornaviruses and related enzymes of como- nepo- and potyviruses. Search of IBV F1 and F2 sequences for sites similar to those cleaved by the latter proteases and intercomparison of the surrounding sequence stretches revealed 13 dipeptides Q/S(G) which are probably cleaved by the coronavirus 3C-like protease. Based on these observations, a partial tentative scheme for the functional organization and expression strategy of the non-structural polyproteins of IBV was proposed. It implies that, despite the general similarity to other positive strand RNA viruses, and particularly to potyviruses, coronaviruses possess a number of unique structural and functional features. PMID:2526320

  7. Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription.

    PubMed Central

    Lemm, J A; Rice, C M

    1993-01-01

    Using vaccinia virus to express Sindbis virus (SIN) nonstructural proteins (nsPs) and template RNAs, we showed previously that synthesis of all three viral RNAs occurred only during expression of either the entire nonstructural coding region or the polyprotein precursors P123 and P34. In this report, the vaccinia virus system was used to express cleavage-defective polyproteins and nsP4 proteins containing various N-terminal extensions to directly examine the roles of the P123 and P34 polyproteins in RNA replication. Replication and subgenomic mRNA transcription occurred during coexpression of P34 and P123 polyproteins in which cleavage was blocked at either or both of the 1/2 and 2/3 sites. For all cleavage-defective P123 polyproteins, however, the ratio of subgenomic to genomic RNA was decreased, suggesting that both the 1/2 and 2/3 cleavages are required for efficient subgenomic RNA transcription. These studies indicate that the uncleaved P123 polyprotein can function as a component of the viral replicase capable of synthesizing both plus- and minus-strand RNAs. In contrast, cleavage-defective P34 was unable to function in RNA replication, even in complementation experiments in which minus-strand RNAs were provided by nsP4. A P34 polyprotein whose cleavage site was not altered could only function in RNA replication in the presence of an active nsP2 protease. Although nsP4, the putative RNA polymerase, was capable of synthesizing only minus-strand RNAs during coexpression with P123, the addition of only 22 upstream residues to nsP4 allowed both replication and transcription of subgenomic RNA to occur. These data show that the conserved domains of both nsP3 and the nsP4 polymerase do not need to be present in a P34 polyprotein to form a functional plus-strand replicase-transcriptase and suggest that the presence of an active nsP2 protease and a cleavable 3/4 site correlates with synthesis of all virus-specific RNA species. Images PMID:8445717

  8. Seismic performance of non-structural components and contents in buildings: an overview of NZ research

    NASA Astrophysics Data System (ADS)

    Dhakal, Rajesh P.; Pourali, Atefeh; Tasligedik, Ali Sahin; Yeow, Trevor; Baird, Andrew; MacRae, Gregory; Pampanin, Stefano; Palermo, Alessandro

    2016-03-01

    This paper summarizes the research on non-structural elements and building contents being conducted at University of Canterbury in New Zealand. Since the 2010-2011 series of Canterbury earthquakes, in which damage to non-structural components and contents contributed heavily to downtime and overall financial loss, attention to seismic performance and design of non-structural components and contents in buildings has increased exponentially in NZ. This has resulted in an increased allocation of resources to research leading to development of more resilient non-structural systems in buildings that would incur substantially less damage and cause little downtime during earthquakes. In the last few years, NZ researchers have made important developments in understanding and improving the seismic performance of secondary building elements such as partitions, facades, ceilings and contents.

  9. Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

    PubMed

    Hwang, Y K; Brinton, M A

    1998-01-01

    The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

  10. Tree Nonstructural Carbohydrate Reserves Across Eastern US Temperate Forests

    NASA Astrophysics Data System (ADS)

    Mantooth, J.; Dietze, M.

    2015-12-01

    Understanding the roles, importance, and dynamics of tree non-structural carbohydrates (NSCs) is currently an active area of research. The question of how the relationships between NSCs, growth, and mortality can be used to develop more accurate projections of forest dynamics is central to this research. To begin to address this question, we have asked an even more fundamental question: How much are trees allocating carbon to storage, in the form of NSCs, versus new growth? Ecological theory predicts that there should be trade-offs between different plant life history strategies provided that there are the carbon mass-balance constraints to enforce these trade-offs. Current data on tree NSCs lack the spatial and taxonomic extent required to properly address this question. Therefore, we established a network of forest inventory plots at ten sites across the eastern US and measured growth in adult trees using increment cores and repeat measures of diameter at breast height (DBH). Increment cores were also used to measure sapwood NSCs. We hypothesized that across the eastern US, shade tolerant species, e.g. Sugar Maple (Acer saccharum) have the largest NSC reserves and that shade intolerant species have the lowest reserves. We also hypothesized that NSC reserves increase with temperature and precipitation, as with growth, and that within species NSC reserves increase with growth rate. Initial analyses of tree NSCs indicates that trees of intermediate shade tolerance, e.g. Red Oak (Quercus rubra) have the highest concentrations of sapwood NSCs, and among the highest growth rates. Across the entire study region, NSC concentrations are positively correlated with tree size and growth rate. Within species, NSC concentrations are also positively correlated with growth rate. Across functional groups healthy individuals have significantly higher sapwood NSC concentrations than visibly stressed individuals. There are also significantly lower NSC concentrations in sapwood of

  11. Response of a 2-story test-bed structure for the seismic evaluation of nonstructural systems

    NASA Astrophysics Data System (ADS)

    Soroushian, Siavash; Maragakis, E. "Manos"; Zaghi, Arash E.; Rahmanishamsi, Esmaeel; Itani, Ahmad M.; Pekcan, Gokhan

    2016-03-01

    A full-scale, two-story, two-by-one bay, steel braced-frame was subjected to a number of unidirectional ground motions using three shake tables at the UNR-NEES site. The test-bed frame was designed to study the seismic performance of nonstructural systems including steel-framed gypsum partition walls, suspended ceilings and fire sprinkler systems. The frame can be configured to perform as an elastic or inelastic system to generate large floor accelerations or large inter story drift, respectively. In this study, the dynamic performance of the linear and nonlinear test-beds was comprehensively studied. The seismic performance of nonstructural systems installed in the linear and nonlinear test-beds were assessed during extreme excitations. In addition, the dynamic interactions of the test-bed and installed nonstructural systems are investigated.

  12. Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives.

    PubMed

    Desta, Seare T; Yuan, XianJun; Li, Junfeng; Shao, Tao

    2016-12-01

    Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility (ED) of Napier grass silage was examined. Napier grass ensiled with no additive control, 0.2% formic acid, 0.4% molasses, and 0.3% fibrolytic enzyme for, 7, 30, 60 and 90days. Additives increased lactic acid, soluble carbohydrate and decreased all of lignocellulosic contents except acid detergent lignin and pH than control. The highest value of nonstructural carbohydrate and large reduction in lignocellulosic contents was observed in formic acid and fibrolytic enzyme silage respectively. The content of glucose and fructose showed rapid drop in the first 7days of ensilage. Ensilage decreased lignocellulosic contents and increased ED compared to fresh material. The ED of formic acid and molasses silage was significantly higher than control and fibrolytic enzyme silages in all tested days. In summery the ensiling quality structural and nonstructural carbohydrate and ED value of mature Napier grass silage improved through additives.

  13. Non-structural carbohydrates in woody plants compared among laboratories.

    PubMed

    Quentin, Audrey G; Pinkard, Elizabeth A; Ryan, Michael G; Tissue, David T; Baggett, L Scott; Adams, Henry D; Maillard, Pascale; Marchand, Jacqueline; Landhäusser, Simon M; Lacointe, André; Gibon, Yves; Anderegg, William R L; Asao, Shinichi; Atkin, Owen K; Bonhomme, Marc; Claye, Caroline; Chow, Pak S; Clément-Vidal, Anne; Davies, Noel W; Dickman, L Turin; Dumbur, Rita; Ellsworth, David S; Falk, Kristen; Galiano, Lucía; Grünzweig, José M; Hartmann, Henrik; Hoch, Günter; Hood, Sharon; Jones, Joanna E; Koike, Takayoshi; Kuhlmann, Iris; Lloret, Francisco; Maestro, Melchor; Mansfield, Shawn D; Martínez-Vilalta, Jordi; Maucourt, Mickael; McDowell, Nathan G; Moing, Annick; Muller, Bertrand; Nebauer, Sergio G; Niinemets, Ülo; Palacio, Sara; Piper, Frida; Raveh, Eran; Richter, Andreas; Rolland, Gaëlle; Rosas, Teresa; Saint Joanis, Brigitte; Sala, Anna; Smith, Renee A; Sterck, Frank; Stinziano, Joseph R; Tobias, Mari; Unda, Faride; Watanabe, Makoto; Way, Danielle A; Weerasinghe, Lasantha K; Wild, Birgit; Wiley, Erin; Woodruff, David R

    2015-11-01

    Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory

  14. Age and Availability of Nonstructural Carbohydrates in Red Maple

    NASA Astrophysics Data System (ADS)

    Carbone, M. S.; Keenan, T. F.; Czimczik, C. I.; Murakami, P.; O'Keefe, J.; Schaberg, P.; Xu, X.; Richardson, A. D.

    2012-12-01

    Recent studies show that nonstructural carbohydrates (NSC) pools in mature trees can be quite large and on average a decade old. Yet, little is known about how older stored NSC reserves vs. recently-assimilated NSCs are used to support growth and metabolism, or how available these stored NSC reserves are to trees during stress or following disturbance. To better understand these aspects of NSC dynamics, we studied mature red maple (Acer rubrum) trees that ranged in size and age in two New England temperate forests, Harvard Forest (Massachusetts) and Bartlett Experimental Forest (New Hampshire). Applying the radiocarbon (14C) "bomb spike" approach, we estimated the age of carbon in stemwood NSCs, bole respiration, and stump sprouts regenerated following harvesting. These isotopic measurements along with stemwood NSC concentrations allowed us to compare the NSC used for metabolic demands and the NSC available for regrowth following disturbance to the NSC actually present in the stemwood. We found that the mean age of stemwood sugars was 9.8 ± 5.3 y. Trees with slower growth rates had older sugar reserves and lower concentrations of sugar, starch, and total NSC reserves. The age of NSCs used to support dormant season metabolism (bole respiration) was between 1-3.5 y, and thus much younger than the mean age of stemwood sugars, indicating preferential use of more recently-assimilated NSC. There were no relationships observed between tree age or size and 1) the age of sugars present in stemwood cores or 2) the age of NSCs used for bole respiration. Moreover, there was no relationship between the age of sugars in stemwood and the age of NSCs used for bole respiration. The stump sprouts were formed from NSCs 1-17 y old, (mean 5.8 ± 5.4 y), with older trees using older NSCs to produce stump sprouts. The stump sprout data indicate that some of these older NSCs reserves are available to the tree for use following major disturbance. However, the bole respiration data

  15. Age, allocation, and availability of nonstructural carbohydrates in red maple

    NASA Astrophysics Data System (ADS)

    Carbone, Mariah; Keenan, Trevor; Czimczik, Claudia; Murakami, Paula; O'Keefe, John; Pederson, Neil; Schaberg, Paul; Xu, Xiaomei; Richardson, Andrew

    2013-04-01

    Nonstructural carbohydrates (NSC) are the primary products of photosynthesis, composed mostly of sugars and starch. Recent studies show that NSC pools in mature trees can be quite large and on average a decade old. Thus, NSC pools integrate years of carbon assimilation and represent significant ecological memory at the whole plant and ecosystem level. However, we know very little about how older stored NSC versus newly assimilated NSC are used to support growth and metabolism, or how available older NSC are to trees during stress or following disturbance. To better understand these potential lags in NSC allocation, we studied mature red maple (Acer rubrum) trees in New England temperate forests. Applying the radiocarbon (14C) "bomb spike" approach, we estimated the age of carbon in stemwood NSC, ring cellulose, bole respiration, and stump sprouts regenerated following harvesting. These measurements allowed us to compare the NSC used for metabolic demands, annual growth, and the NSC available for regrowth following disturbance to the NSC actually present in the stemwood. Finally, tree ring widths were analyzed to determine the annual autocorrelation in radial wood increment. We found that the mean age of stemwood sugars was 9.8 ± 5 y. The age of NSC used to support metabolism (bole respiration) was much younger than the mean age of stemwood sugars, indicating preferential use of more recently assimilated NSC. In the spring before leaves emerged, bole respiration was between 1-2 y, whereas it was composed of newly assimilated NSC in the late summer. The ring cellulose 14C age was on average 0.8 y older than direct ring counts (within error of 14C measurement) which may or may not indicate a stored NSC contribution. Tree ring width analyses indicate strong autocorrelation between ring growth in one year and in the following year, in agreement with ring cellulose 14C ages. However, autocorrelation weakened over the following 10 years, consistent with the measured mean

  16. Modeling nonstructural carbohydrate reserve dynamics in forest trees

    NASA Astrophysics Data System (ADS)

    Richardson, A. D.; Keenan, T. F.; Carbone, M. S.; Czimczik, C. I.; Hollinger, D. Y.; Murakami, P.; Schaberg, P.; Xu, X.

    2012-12-01

    Understanding the factors influencing the availability of nonstructural carbohydrate (NSC) reserves is essential for predicting the resilience of forests to climate change and environmental stress. However, carbon allocation processes remain poorly understood and many models either ignore NSC reserves, or use simple and untested representations of NSC allocation and pool dynamics. Using model-data fusion techniques, we combined a parsimonious model of forest ecosystem carbon cycling with novel field sampling and laboratory analyses of NSCs. Simulations were conducted for an evergreen conifer forest and a deciduous broadleaf forest in New England. We used radiocarbon methods based on the 14C "bomb spike" to estimate the age of NSC reserves, and used this to constrain the mean residence time of modeled NSCs. We used additional data, including tower-measured fluxes of CO2, soil and biomass carbon stocks, woody biomass increment, and leaf area index and litterfall, to further constrain the model's parameters and initial conditions. Three years of field measurements indicate that stemwood NSCs are highly dynamic on seasonal time scales. The modeled seasonal dynamics conform to expectations (accumulated in the growing season, depleted in the dormant season) but are inconsistent with the observational data (total stemwood NSC concentrations higher in March than November, lower in August than June). We interpret this contradiction to suggest that stemwood concentrations provide an incomplete picture of the whole-tree NSC budget. A two-pool model structure that accounted for both "fast" (active pool, MRT ≈1 y) and "slow" (passive pool, MRT ≥ 20 y) cycling reserves (1) gives reasonable estimates of the size and MRT of the total NSC pool; (2) greatly improves model predictions of interannual variability in woody biomass increment, compared to zero- or one-pool structures used in the majority of existing models; (3) provides a mechanism by which observations of a one

  17. Seasonal changes of C and N non-structural compounds in the stem sapwood of adult sessile oak and beech trees.

    PubMed

    El Zein, R; Maillard, P; Bréda, N; Marchand, J; Montpied, P; Gérant, D

    2011-08-01

    We assessed the pools of non-structural nitrogen compounds (NSNC) through a year, thereby addressing the question of whether mature sessile oak [Quercus petraea (Matt.) Liebl.] and beech (Fagus sylvatica L.), which differ in wood anatomy and growth patterns, exhibit contrasting seasonal dynamics of NSNC pools as previously shown for non-structural carbohydrate (NSC) pools. Seasonal fluctuations of NSNC (amino acids and soluble proteins) and NSC (starch and soluble sugars) pools were analyzed in the inner and the outer stem sapwood. In oak, NSC showed marked seasonal variation within the stem sapwood (accumulation during winter and decrease during bud burst and early wood growth), whereas in beech seasonal fluctuations in NSC were of minor amplitude. Even if the distribution and intensity of the NSNC pools differed between the two species, NSNC of the stem sapwood did not show seasonal variation. The most significant change in NSNC pools was the seasonal fluctuation of protein composition. In both species, two polypeptides of 13 kDa (PP13) and 26 kDa (PP26) accumulated during the coldest period in parallel with starch to sugar conversion and disappeared with the onset of spring growth. The absence of seasonal changes in total soluble protein concentration suggests that the polypeptides are involved in the internal nitrogen (N) cycling of the stem rather than in N storage and remobilization to the other growing organs of the tree.

  18. 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

    SciTech Connect

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    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 indicated 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.

  19. Seasonal dynamics and age of stemwood nonstructural carbohydrates in temperate forest trees.

    PubMed

    Richardson, Andrew D; Carbone, Mariah S; Keenan, Trevor F; Czimczik, Claudia I; Hollinger, David Y; Murakami, Paula; Schaberg, Paul G; Xu, Xiaomei

    2013-02-01

    Nonstructural carbohydrate reserves support tree metabolism and growth when current photosynthates are insufficient, offering resilience in times of stress. We monitored stemwood nonstructural carbohydrate (starch and sugars) concentrations of the dominant tree species at three sites in the northeastern United States. We estimated the mean age of the starch and sugars in a subset of trees using the radiocarbon ((14) C) bomb spike. With these data, we then tested different carbon (C) allocation schemes in a process-based model of forest C cycling. We found that the nonstructural carbohydrates are both highly dynamic and about a decade old. Seasonal dynamics in starch (two to four times higher in the growing season, lower in the dormant season) mirrored those of sugars. Radiocarbon-based estimates indicated that the mean age of the starch and sugars in red maple (Acer rubrum) was 7-14 yr. A two-pool (fast and slow cycling reserves) model structure gave reasonable estimates of the size and mean residence time of the total NSC pool, and greatly improved model predictions of interannual variability in woody biomass increment, compared with zero- or one-pool structures used in the majority of existing models. This highlights the importance of nonstructural carbohydrates in the context of forest ecosystem carbon cycling.

  20. Interspecies variation of forage nutritive value and nonstructural carbohydrates in perennial cool-season grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forage nitrogen (N) and nonstructural carbohydrate (NSC) concentrations are important indicators of forage quality, and knowledge of N and NSC variation among grass germplasm is one element to consider in developing a successful forage and livestock management program. An experiment was conducted in...

  1. Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in trans

    PubMed Central

    Grassmann, Claus W.; Isken, Olaf; Tautz, Norbert; Behrens, Sven-Erik

    2001-01-01

    The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of

  2. Locations of nonstructural hydrocarbon traps on the shelf of the northern Barents Sea

    NASA Astrophysics Data System (ADS)

    Kazanin, G. S.; Pavlov, S. P.; Tarasov, G. A.; Schlykova, V. V.; Matishov, G. G.

    2016-11-01

    This paper considers the results of summarized integrated geophysical investigations that were carried out from 2006 to 2012. The investigations included common depth point (CDP) seismic reflection survey, over water gravity survey, and differential hydromagnetic exploration with a total work scope of 30 000 linear kilometers. The deep structural tectonic plan, the structural and lithofacies features of the sedimentary cover section on the basic reflecting boundaries, and the features of the seismogeological complexes and seismic sections on a depth scale have been studied, and geological oil-and-gas zoning of the Northern Barents shelf has been made. Seventy-nine local anticlinal highs have been revealed, and the zones with potential nonstructural hydrocarbon traps have been determined. Due to the lack of huge anticlinal highs in the northern Barents Sea region, nonstructural traps are of interest in studying and replacing the mineral raw material base of Russia, as well as for arranging marine exploration.

  3. Direct Replacement of Arbitrary Grid-Overlapping by Non-Structured Grid

    NASA Technical Reports Server (NTRS)

    Kao, Kai-Hsiung; Liou, Meng-Sing

    1994-01-01

    A new approach that uses nonstructured mesh to replace the arbitrarily overlapped structured regions of embedded grids is presented. The present methodology uses the Chimera composite overlapping mesh system so that the physical domain of the flowfield is subdivided into regions which can accommodate easily-generated grid for complex configuration. In addition, a Delaunay triangulation technique generates nonstructured triangular mesh which wraps over the interconnecting region of embedded grids. It is designed that the present approach, termed DRAGON grid, has three important advantages: eliminating some difficulties of the Chimera scheme, such as the orphan points and/or bad quality of interpolation stencils; making grid communication in a fully conservative way; and implementation into three dimensions is straightforward. A computer code based on a time accurate, finite volume, high resolution scheme for solving the compressible Navier-Stokes equations has been further developed to include both the Chimera overset grid and the nonstructured mesh schemes. For steady state problems, the local time stepping accelerates convergence based on a Courant - Friedrichs - Leury (CFL) number near the local stability limit. Numerical tests on representative steady and unsteady supersonic inviscid flows with strong shock waves are demonstrated.

  4. Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever.

    PubMed

    Huang, J H; Wey, J J; Sun, Y C; Chin, C; Chien, L J; Wu, Y C

    1999-01-01

    Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1-15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection.

  5. The KnowRISK project - Know your city, Reduce seISmic risK through non-structural elements

    NASA Astrophysics Data System (ADS)

    Sousa Oliveria, Carlos; Amaral Ferreira, Mónica; Lopez, Mário; Sousa Silva, Delta; Musacchio, Gemma; Rupakhety, Rajesh; Falsaperla, Susanna; Meroni, Fabrizio; Langer, Horst

    2016-04-01

    Historically, there is a tendency to focus on seismic structural performance of buildings, neglecting the potential for damage of non-structural elements. In particular, non-structural elements of buildings are their architectural parts (i.e. partitions, ceilings, cladding), electrical and mechanical components (i.e., distribution panels, piping, plumbing), and contents (e.g., furniture, bookcases, computers and desktop equipment). Damage of these elements often contributes significantly to earthquake impacts. In the 1999 Izmit Earthquake, Turkey, 50% of the injuries and 3% of human losses were caused by non-structural failures. In the 2010-2011 Christchurch Earthquakes (New Zealand), 40% of building damage was induced by non-structural malfunctions. Around 70%-85% of construction cost goes into these elements, and their damage can strongly influence the ability of communities to cope with and recover from earthquakes. The project Know your city, Reduce seISmic risK through non-structural elements (KnowRISK) aims at facilitating local communities' access to expert knowledge on non-structural seismic protection solutions. The project will study seismic scenarios critical for non-structural damage, produce a portfolio of non-structural protection measures and investigate the level of awareness in specific communities. We will implement risk communication strategies that will take into account the social and cultural background and a participatory approach to raise awareness in local communities. The paradox between the progress of scientific knowledge and the ongoing increase of losses from natural disasters worldwide is a well-identified gap in the UN Hyogo Framework for Action 2005-2015, in which one of the main priorities is the investment on "knowledge use, innovation and education to build a culture of safety and resilience". The KnowRISK is well aligned with these priorities and will contribute to participatory action aimed at: i) transferring expert knowledge

  6. Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients.

    PubMed

    Yoshida, C F; Takahashi, Y; Vanderborght, B O; Rouzere, C D; França, M S; Takahashi, C; Takamizawa, A; Yoshida, I; Schatzmayr, H G

    1993-01-01

    Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

  7. Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses

    PubMed Central

    Holmström, Fredrik; Chen, Margaret; Balasiddaiah, Anangi; Sällberg, Matti; Ahlén, Gustaf; Frelin, Lars

    2016-01-01

    The hepatitis C virus nonstructural (NS) 3/4A and NS5A proteins are major targets for the new direct-acting antiviral compounds. Both viral proteins have been suggested as modulators of the response to the host cell. We have shown that NS3/4A- and NS5A-specific T cell receptors confer different effector functions, and that killing of NS3/4A-expressing hepatocytes is highly dependent on IFN-γ. We here characterize the functional differences in the T cell responses to NS3/4A and NS5A. NS3/4A- and NS5A-specific T cells could be induced at various frequencies in wild-type-, NS3/4A-, and NS5A-transgenic mice. Priming of NS5A-specific T cells required a high DNA dose, and was unlike NS3/4A dependent on both CD4+ and CD8+ T cells, but less influenced by CD25+/GITR+ regulatory T cells. The presence of IL-12 greatly improved specific CD8+ T cell priming by NS3/4A but not by NS5A, suggesting a less dependence of IFN-γ for NS5A. This notion was supported by the observation that NS5A-specific T cells could eliminate NS5A-expressing hepatocytes also in the absence of IFN-γ-receptor-2. This supports that NS3/4A- and NS5A-specific T cells become activated and eliminate antigen expressing, or infected hepatocytes, by distinct mechanisms, and that NS5A-specific T cells show an overall less dependence of IFN-γ. PMID:27141891

  8. [Genetic analysis of the nonstructural gene (NS1) of H9N2 avian influenza viruses isolated in China].

    PubMed

    Li, Jian-Li; Chen, En-Lin; Li, He-Ping; Hao, Chun-Li; Bi, Ding-Ren; Wang, Ze-Lin

    2008-05-01

    The nonstructural genes (NS1) of nine H9N2 subtype avian influenza viruses isolated from diseased chickens on different farms during 1998-2005 were amplified by RT-PCR and completely sequenced. The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were compared. The results showed that NS1 genes of all H9N2 isolates contained 654 bp and encoded 217 amino acids. The homologies of the nucleotide and deduced amino sequences of the isolates were 95.4%-99.8% and 93.6%-100%, respectively. Comparison of the amino acid sequences of NS1 proteins of these isolates with other H9N2 viruses demonstrated that NS1 proteins of the nine strains had a deletion of 13 amino acid residues at the carboxyl terminus, which may be the molecule mark of the isolates in mainland China. Phylogenetic analysis revealed that the NS1 genes of these isolates fell into the same lineage and belonged to allele A. Eight out of nine isolates belonged to the CK/SH/F/98-like lineage while only Ck/HN/A3/98 strain belonged to the Ck/HK/Y280/97-like linease. All the isolates were derived from Ck/BJ/1/94 strain which was the first isolate is mainland China in 1994. The results indicated that H9N2 subtype AIV appeared differentiation following the passage of time and the viruses belonging to Ck/SH/F/98-like acquired an epidemic spread advantage in chicken population in mainland China.

  9. The role of non-structural carbohydrate reserves in trees under climatic stress

    NASA Astrophysics Data System (ADS)

    Hoch, G.; Koerner, C.

    2012-12-01

    The storage of non-structural carbon (C) reserves is an indispensable process for all plants. Diverting parts of their photoassimilates into storage pools (e.g. starch and storage lipids) ensures plants to survive periods when the requirement for C (i.e. the sum of all C-sink activities) exceeds their photosynthetic capacity (C-source activity). Over the last decade, research delivered clear evidence that under the current atmospheric CO2 concentrations, tree growth is generally limited by the availability of resources other than C (e.g. soil nutrients) under most conditions. Whether climatic stresses, like cold temperature or drought, can induce C-limitation in trees is currently vividly debated. We will thus address the following questions: 1) do low temperatures at alpine treelines or drought lead to situations where photosynthesis is limiting growth, and 2) what is the role of non-structural C reserves under such conditions? Trees at the alpine treeline as well as under hydraulic constraints accumulate, rather than use up their non-structural carbohydrate reserves with increasing stress. We propose that this observed increase of C stores results from a stress related decline in growth (C-sink activity) relative to C-supply. Hence, the higher C reserve concentrations found in trees under cold and dry conditions are very likely a direct physiological response to the environmental stress (diversion to storage), reflecting relatively higher availability of photoassimilates compared to sink demand. Previous experiments with trees and crops showed that in cold and dry environments, meristematic growth is generally limited more severely and at an earlier stage than photosynthesis. While cell division and differentiation are close to zero at about 5 °C even in cold adapted species, rates of photosynthesis are still reaching up to 80 % of maximum rates. Similarly, structural growth generally ceases at much less negative water potentials than does photosynthesis, which

  10. Effect of non-structural elements on the dynamic behaviour of moment-resisting framed structures

    NASA Astrophysics Data System (ADS)

    Carlo Ponzo, Felice; Ditommaso, Rocco; Auletta, Gianluca

    2015-04-01

    Effects of earthquakes on building structures have studied from many researchers on the recent scientific and technique literature. The phenomenon is clear: inertia forces are governed from structural and non-structural stiffness and masses. The distribution of seismic lateral loads and their magnitude are strongly correlated to the fundamental period of the structure. Therefore, an accurate evaluation of the fundamental period is a crucial aspect for both static and dynamic seismic analyses. In fact, the fundamental period determines the global seismic demand through the spectral acceleration directly evaluated from the linear and/or nonlinear acceleration response spectra (provided from codes or derived from detailed analyses of site effects). Recent earthquakes highlighted the significant effects derived from the interaction between structural and non-structural elements on the main dynamic parameters of a structure and on the lateral distribution of the inertial forces. Usually, non-structural elements acts together with the structural elements, adding both masses and stiffness. Using numerical and experimental campaigns, many researchers have studied the effects of infill walls on the dynamic behaviour of buildings and several simplified models have been proposed to take into account the presence of non-structural elements within linear and nonlinear numerical models. As example, Kliner and Bertero tested a 1/3 scaled structure (moment-resisting infilled frame model) and determine its behaviour during earthquakes. They found that the infills increased the stiffness of the frame in about 5 times. Consequently, in these cases the fundamental period reduces and the inertia forces generally increases. Meharabi et al. tested a 6-storey, three bay, reinforced concrete moment resisting frame, designed according to the provision of UBC-91, and they shown that the lateral force resistance of an infilled frame was higher than that of bare frame. It was concluded that a

  11. Logical Schema Acquisition from Text-Based Sources for Structured and Non-Structured Biomedical Sources Integration

    PubMed Central

    García-Remesal, Miguel; Maojo, Víctor; Crespo, José; Billhardt, Holger

    2007-01-01

    In this paper we present a novel approach to integrate non-structured and structured sources of biomedical information. We part from previous research on database integration conducted in the context of the EC funded INFOGENMED project. In this project we developed the ONTOFUSION system, which provides a robust framework to integrate large sets of structured biomedical sources. Methods and tools provided by ONTOFUSION cannot be used to integrate non-structured sources, since the latter usually lack a logical schema. In this article we introduce a novel method to extract logical schemas from text-based collections of biomedical information. Non-structured sources equipped with a logical schema can be regarded as regular structured sources, and thus can be bridged together using the methods and tools provided by ONTOFUSION. To test the validity of this approach, we carried out an experiment with a set of five cancer databases. PMID:18693838

  12. RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins.

    PubMed

    Kallio, Katri; Hellström, Kirsi; Jokitalo, Eija; Ahola, Tero

    2015-11-18

    The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

  13. RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins

    PubMed Central

    Kallio, Katri; Hellström, Kirsi; Jokitalo, Eija

    2015-01-01

    The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected. PMID:26581991

  14. Coronavirus non-structural protein 16: Evasion, attenuation, and possible treatments

    PubMed Central

    Menachery, Vineet D.; Debbink, Kari; Baric, Ralph S.

    2014-01-01

    The recent emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), nearly a decade after the Severe Acute Respiratory Syndrome (SARS) CoV, highlights the importance of understanding and developing therapeutic treatment for current and emergent CoVs. This manuscript explores the role of NSP16, a 2′O-methyl-transferase (2′O-MTase), in CoV infection and the host immune response. The review highlights conserved motifs, required interaction partners, as well as the attenuation of NSP16 mutants, and restoration of these mutants in specific immune knockouts. Importantly, the work also identifies a number of approaches to exploit this understanding for therapeutic treatment and the data clearly illustrate the importance of NSP16 2′O-MTase activity for CoV infection and pathogenesis. PMID:25278144

  15. Automatic procedure exploiting multicommutation in flow analysis for simultaneous spectrophotometric determination of nonstructural carbohydrates and reducing sugar in forage materials.

    PubMed

    Tumang, Cristiane A; Tomazzini, Maria Cristina; Reis, Boaventura F

    2003-12-01

    A multicommutated flow procedure for simultaneous spectrophotometric determination of nonstructural carbohydrates and reducing sugar in forage materials is proposed. Determination of both analytes was based on the neucroine method after acid hydrolysis of nonstructural carbohydrates. Results for both analytes presented the following features: no significant difference at 90% confidence level when compared with reference method, linear response between 0.2 to 0.8% (w/v) (r = 0.999); relative standard deviations of < 2.0% (n = 10), and sampling rate of 32 determinations per hour.

  16. Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice.

    PubMed

    Wang, Diane R; Wolfrum, Edward J; Virk, Parminder; Ismail, Abdelbagi; Greenberg, Anthony J; McCouch, Susan R

    2016-11-01

    Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. We find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice.

  17. Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

    PubMed Central

    Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder; Ismail, Abdelbagi; Greenberg, Anthony J.; McCouch, Susan R.

    2016-01-01

    Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. We find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice. PMID:27707775

  18. Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

    DOE PAGES

    Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder; ...

    2016-10-05

    Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established anmore » appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. Lastly, we find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice.« less

  19. Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

    SciTech Connect

    Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder; Ismail, Abdelbagi; Greenberg, Anthony J.; McCouch, Susan R.

    2016-10-05

    Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. Lastly, we find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice.

  20. Artificial defoliation effect on Populus growth, biomass production, and total nonstructural carbohydrate concentration

    SciTech Connect

    Reichenbacker, R.R.; Hart, E.R.; Schultz, R.C.

    1996-06-01

    The impact of artificial defoliation on Populus growth, biomass production, and total nonstructural carbohydrate concentration was examined. Four Populus clones were field planted and artificially defoliated. Assigned defoliation levels (0, 25, 50, or 75%) were applied to leaves of leaf plastochron index 0 through 8 during a 6-d period in a 3-step incremental manner to simulate cottonwood leaf beetle, Chrysomela scripta F., larval feeding patterns. Artificial defoliations were timed to coincide with the outbreaks of natural beetle populations in adjacent areas. After 2 growing seasons, trees were measured for height, diameter, and biomass accumulation. Root samples were collected from 0 and 75% defoliation treatments for each clone. Biomass was reduced an average of 33% as defoliation level increased from 0 to 75%. As defoliation level increased from 0 to 75%, a consistent allocation ratio of biomass to 2/3 above and 1/3 below ground components continued in all clones. An overcompensation response occurred in above ground biomass when a defoliation level of 25% was applied. Between 25 and 75% a strong linear trend of decreasing biomass as defoliation increased was indicated. Vitality of the tree, as indicated by total nonstructural carbohydrate content, was affected only slightly by increasing defoliation. 26 refs., 1 fig., 6 tabs.

  1. Functional assignment to JEV proteins using SVM

    PubMed Central

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

  2. Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes.

    PubMed

    Posthuma, Clara C; Te Velthuis, Aartjan J W; Snijder, Eric J

    2017-02-06

    Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26-34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3'-to-5' exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.

  3. Genetic effects on total phenolics, condensed tannins and non-structural carbohydrates in loblolly pine (Pinus taeda L.) needles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic and environmental effects on carbon allocation to soluble phenolics and non-structural carbohydrates in needles of widely-planted loblolly pine (Pinus taeda L.) genotypes could impact productivity, sustainability and biogeochemical cycling in the southeastern U.S. The magnitude of genetic a...

  4. Profiles of Nonstructural Carbohydrates, as a Function of Species and Extraction Method, in Four Cool-Season Forage Grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nonstructural carbohydrates (NSC) of forage grasses, particularly long-chain fructans, have been proposed as the causal agent of equine laminitis. In order to evaluate the correlation between NSC and laminitis, NSC must be quantified in the forages being fed to and grazed by horses. The goal of th...

  5. Quantitative characterization of nonstructural carbohydrates of mezcal Agave (Agave salmiana Otto ex Salm-Dick).

    PubMed

    Michel-Cuello, Christian; Juárez-Flores, Bertha Irene; Aguirre-Rivera, Juan Rogelio; Pinos-Rodríguez, Juan Manuel

    2008-07-23

    Fructans are the reserve carbohydrates in Agave spp. plants. In mezcal factories, fructans undergoes thermal hydrolysis to release fructose and glucose, which are the basis to produce this spirit. Carbohydrate content determines the yield of the final product, which depends on plant organ, ripeness stage, and thermal hydrolysis. Thus, a qualitative and quantitative characterization of nonstructural carbohydrates was conducted in raw and hydrolyzed juices extracted from Agave salmiana stems and leaves under three ripeness stages. By high-performance liquid chromatography (HPLC), fructose, glucose, sucrose, xylose, and maltose were identified in agave juice. Only the plant fraction with hydrolysis interaction was found to be significant in the glucose concentration plant. Interactions of the fraction with hydrolysis and ripeness with hydrolysis were statistically significant in fructose concentration. Fructose concentration rose considerably with hydrolysis, but only in juice extracted from ripe agave stems (early mature and castrated). This increase was statistically significant only with acid hydrolysis.

  6. Nonstructural heart disease in the newborn. Observations during one year in a perinatal service.

    PubMed Central

    Rowe, R D; Izukawa, T; Mulholland, H C; Bloom, K R; Cook, D H; Swyer, P R

    1978-01-01

    One-third of 327 newborn infants referred to the perinatal service of the Hospital for Sick Children during 1975 with suspected cardiopulmonary disorders proved to have nonstructural heart disease. Most of these were term infants with transient tachypnoea or cyanosis who recovered. A history of fetal distress or difficult delivery was commonly associated. The haemodynamic disorder for most was a delay in the normal progress of the transitional circulation. Evidence of myocardial ischaemia was present in 40%, and about half of these developed congestive heart failure. Aids to diagnosis of the ischaemic complication included echocardiography and myocardial perfusion scanning. For a small proportion specific metabolic disturbances, myocarditis, or dysrhythmia seemed the primary cause but even for these there were reasonable grounds to suspect a prenatal origin. Current general supportive measures were of value in treatment. PMID:718241

  7. The effect of non-structural components and lignin on hemicellulose extraction.

    PubMed

    Liu, Kai-Xuan; Li, Hong-Qiang; Zhang, Jie; Zhang, Zhi-Guo; Xu, Jian

    2016-08-01

    As the important structural component of corn stover, hemicellulose could be converted into a variety of high value-added products. However, high quality hemicellulose extraction is not an easy issue. The present study aims to investigate the effects of non-structural components (NSCs) and lignin removal on alkaline extraction of hemicellulose. Although NSCs were found to have a minimal effect on hemicellulose dissolution, they affected the color values of the hemicellulose extracts. The lignin limited the hemicellulose dissolution and increased the color value by binding to hemicellulose molecules and forming lignin-carbohydrate complexes. Sodium chlorite method can remove about 90% lignin from corn stover, especially the lignin connected to hemicellulose through p-coumaric and ferulic acids. Which increased the hemicellulose dissolution ratio to 93% and reduced the color value 14-28%, but the cost is about 20% carbohydrates lost.

  8. Effects of ozone and water deficit on field-grown soybean: II. Leaflet nonstructural carbohydrates

    SciTech Connect

    Miller, J.E.; Vozzo, S.F.; Patterson, R.P.

    1995-07-01

    Ozone (O{sub 3}) and water deficit can suppress photosynthesis, growth, and yield of crops, and both may alter plant carbohydrate status. Little is known, however, concerning the combined effects of these stresses on C assimilation and nonstructural carbohydrate reserves in field-grown plants. Soybean [Glycine max (L.) Merr. `Young`] plants were subjected to two soil moisture regimes (providing well-watered and periodically water-deficient conditions) and three levels of O{sub 3} concentrations were 0.018, 0.059, and 0.085 {mu}L L{sup -1} (seasonal mean 12 h d{sup -1} concentration). Leaflet carbohydrate concentrations were measured periodically during the growing season. Total soluble carbohydrates (TSCs) (sucrose and hexose) and starch were measured in the center leaflet of the sixth trifoliolate from the apex. Ozone stress suppressed leaflet concentrations of TSCs and starch on most sampling dates. Impacts of water deficit were less consistent, but starch concentrations usually increased when effects were significant. Interactions between the two stresses occurred infrequently, although water stress reduced the negative effects of O{sub 3} on sucrose and TSCs when the data were analyzed over the season. Ozone treatment also slightly increased the proportion of sucrose compared to starch in the total nonstructural carbohydrate (TNC) pool. The response of seasonal mean TNC concentrations, seasonal mean NCER, and seed yield to O{sub 3} followed similar patterns, although TNCs were suppressed more on a relative basis then NCER or yield. 34 refs., 4 figs., 4 tabs.

  9. [Spatial variation of non-structural carbohydrates in Betula platyphylla and Tilia amurensis stems].

    PubMed

    Zhang, Hai-Yan; Wang, Chuan-Kuan; Wang, Xing-Chang; Cheng, Fang-Yan

    2013-11-01

    Taking the two diffuse-porous tree species Betula platyphylla and Tilia amurensis in a temperate forest in Northeast China as test objects, this paper studied the spatial variation of the non-structural carbohydrates (NSC) concentrations in the stem xylem after leaf-fall. For the two tree species, the concentrations of total non-structural carbohydrate (TNC, soluble sugars plus starch) and soluble sugars in the stem xylem decreased gradually with the increasing depth from cambium to pith, whereas the starch concentration showed little radial variation. There was still a substantial amount of NSC in the inner wood close to pith. The concentrations of the NSC in the two species stems decreased gradually from the stump to the breast height, and then increased vertically. The maximum concentrations of the TNC, soluble sugars, and starch occurred at different heights, depending on the species and the TNC components. The ratio of sugar to starch showed a contrasting vertical trend for the two species, i. e., increasing from the stump to the top for B. platyphylla, but decreasing for T. amurensis. The estimation error of the stem NSC storage was mainly from the axial variation, and then, from the radial variation of NSC concentration. The TNC concentration (1.0% dry mass) in the stem of shade-intolerant species B. platyphylla was significantly lower than that (4.3% dry mass) of shade-tolerant species T. amurensis, which could be related to their different life-history strategies. Applying the sampling protocols considering the axial and radial variations of NSC could effectively reduce the potential uncertainty in estimating the NSC storage at tree or stand level.

  10. Mutations in the classical swine fever virus NS4B protein affects virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NS4B is one of the non-structural proteins of Classical Swine Fever Virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a Toll/Inte...

  11. Development and characterization of mouse monoclonal antibodies against monomeric dengue virus non-structural glycoprotein 1 (NS1).

    PubMed

    Gelanew, Tesfaye; Poole-Smith, B Katherine; Hunsperger, Elizabeth

    2015-09-15

    Dengue virus (DENV) nonstructural-1 (NS1) glycoprotein is useful for diagnosis of DENV infections in the first 8 days of illness with any of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). However, NS1 diagnostics are less sensitive for secondary DENV infections so the utility of NS1 diagnostics in dengue endemic countries where there is predominantly secondary infections is being questioned. Heat-mediated immunecomplex dissociation (ICD) prior to testing serum samples can significantly improve NS1 test sensitivity in secondary infections but requires monoclonal antibodies (MAbs) reactive to heat-denatured NS1. In order to incorporate a simple heat-mediated ICD step, a crucial step was to develop new MAbs with high affinity and specificity to heat-denatured DENV NS1 protein. In the present study, six new MAbs were isolated from BALB/c mice immunized with recombinant monomeric NS1 of DENV-1 and DENV-2. Characterization using three different methods: indirect ELISA, fixed cell ELISA and western blot revealed that all six MAbs are serotype-cross-reactive and capable of recognizing dimeric and hexameric isoforms as well as heat-denatured NS1 from all four DENV serotypes. No cross-reactivity to NS1 of West Nile virus and Yellow fever virus was observed on western blot and indirect ELISA. Five of the six MAbs mapped to the DENV NS1 region of 105-119 amino acids. The remaining MAb mapped to DENV NS1 region of 25-39 amino acids. These two NS1 regions were found to be highly conserved among all four DENV serotypes by sequences analysis and database comparison. These MAbs were used to develop an NS1 capture ELISA and tested using a small panel of clinical specimens. The results from the NS1 capture ELISA indicated at least a three-fold increase in NS1 antigen detection in heat-denatured samples compared to untreated specimens. Furthermore, artificial immunecomplexed results also demonstrated the binding efficiency of these MAbs to heat denatured NS1. Taken together

  12. Wind-Tunnel Evaluation of the Effect of Blade Nonstructural Mass Distribution on Helicopter Fixed-System Loads

    NASA Technical Reports Server (NTRS)

    Wilbur, Matthew L.; Yeager, William T., Jr.; Singleton, Jeffrey D.; Mirick, Paul H.; Wilkie, W. Keats

    1998-01-01

    This report provides data obtained during a wind-tunnel test conducted to investigate parametrically the effect of blade nonstructural mass on helicopter fixed-system vibratory loads. The data were obtained with aeroelastically scaled model rotor blades that allowed for the addition of concentrated nonstructural masses at multiple locations along the blade radius. Testing was conducted for advance ratios ranging from 0.10 to 0.35 for 10 blade-mass configurations. Three thrust levels were obtained at representative full-scale shaft angles for each blade-mass configuration. This report provides the fixed-system forces and moments measured during testing. The comprehensive database obtained is well-suited for use in correlation and development of advanced rotorcraft analyses.

  13. Solidification/stabilization of spent abrasives and use as nonstructural concrete

    SciTech Connect

    Brabrand, D.J.; Loehr, R.C. )

    1993-01-01

    Tons of spent abrasives result each year from the removal of old paint from bridges. Because the spent abrasives contain metals from the paint, some spent abrasives may be considered hazardous by the Toxicity Characteristic (TC) criteria. Incorporation of the spent blasting abrasives in nonstructural concrete (rip-rap, dolphins) offers an opportunity to recycle the spent abrasives while immobilizing potentially leachable metals. This study focused on the Portland Cement Solidification/Stabilization (S/S) of spent blasting abrasives taken from a bridge located in Southeast Texas. The study examined (a) the cadmium, chromium, and lead concentrations in extracts obtained by using the Toxicity Characteristic Leaching Procedure (TCLP) and (b) the compressive strengths of Portland Cement mixes that contained different amounts of the spent abrasives. Performance was measured by meeting the TC criteria as well as the requirements for compressive strength. Study results indicated that considerable quantities of these spent abrasives can be solidified/stabilized while reducing the leachability of cadmium, chromium, and lead and producing compressive strengths over 6,895 kN/m[sup 2] (1,000 psi).

  14. Distribution and mixing of old and new nonstructural carbon in two temperate trees

    PubMed Central

    Richardson, Andrew D; Carbone, Mariah S; Huggett, Brett A; Furze, Morgan E; Czimczik, Claudia I; Walker, Jennifer C; Xu, Xiaomei; Schaberg, Paul G; Murakami, Paula

    2015-01-01

    We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two temperate trees, and we used the continuous label offered by the radiocarbon (carbon-14, 14C) bomb spike to estimate the mean age of NSC in different tissues. NSC in branches and the outermost stemwood growth rings had the 14C signature of the current growing season. However, NSC in older aboveground and belowground tissues was enriched in 14C, indicating that it was produced from older assimilates. Radial patterns of 14C in stemwood NSC showed strong mixing of NSC across the youngest growth rings, with limited ‘mixing in’ of younger NSC to older rings. Sugars in the outermost five growth rings, accounting for two-thirds of the stemwood pool, had a mean age < 1 yr, whereas sugars in older growth rings had a mean age > 5 yr. Our results are thus consistent with a previously-hypothesized two-pool (‘fast’ and ‘slow’ cycling NSC) model structure. These pools appear to be physically distinct. PMID:25558814

  15. Distribution and mixing of old and new nonstructural carbon in two temperate trees.

    PubMed

    Richardson, Andrew D; Carbone, Mariah S; Huggett, Brett A; Furze, Morgan E; Czimczik, Claudia I; Walker, Jennifer C; Xu, Xiaomei; Schaberg, Paul G; Murakami, Paula

    2015-04-01

    We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two temperate trees, and we used the continuous label offered by the radiocarbon (carbon-14, (14) C) bomb spike to estimate the mean age of NSC in different tissues. NSC in branches and the outermost stemwood growth rings had the (14) C signature of the current growing season. However, NSC in older aboveground and belowground tissues was enriched in (14) C, indicating that it was produced from older assimilates. Radial patterns of (14) C in stemwood NSC showed strong mixing of NSC across the youngest growth rings, with limited 'mixing in' of younger NSC to older rings. Sugars in the outermost five growth rings, accounting for two-thirds of the stemwood pool, had a mean age < 1 yr, whereas sugars in older growth rings had a mean age > 5 yr. Our results are thus consistent with a previously-hypothesized two-pool ('fast' and 'slow' cycling NSC) model structure. These pools appear to be physically distinct.

  16. Age, allocation and availability of nonstructural carbon in mature red maple trees.

    PubMed

    Carbone, Mariah S; Czimczik, Claudia I; Keenan, Trevor F; Murakami, Paula F; Pederson, Neil; Schaberg, Paul G; Xu, Xiaomei; Richardson, Andrew D

    2013-12-01

    The allocation of nonstructural carbon (NSC) to growth, metabolism and storage remains poorly understood, but is critical for the prediction of stress tolerance and mortality. We used the radiocarbon ((14) C) 'bomb spike' as a tracer of substrate and age of carbon in stemwood NSC, CO2 emitted by stems, tree ring cellulose and stump sprouts regenerated following harvesting in mature red maple trees. We addressed the following questions: which factors influence the age of stemwood NSC?; to what extent is stored vs new NSC used for metabolism and growth?; and, is older, stored NSC available for use? The mean age of extracted stemwood NSC was 10 yr. More vigorous trees had both larger and younger stemwood NSC pools. NSC used to support metabolism (stem CO2 ) was 1-2 yr old in spring before leaves emerged, but reflected current-year photosynthetic products in late summer. The tree ring cellulose (14) C age was 0.9 yr older than direct ring counts. Stump sprouts were formed from NSC up to 17 yr old. Thus, younger NSC is preferentially used for growth and day-to-day metabolic demands. More recently stored NSC contributes to annual ring growth and metabolism in the dormant season, yet decade-old and older NSC is accessible for regrowth.

  17. Drought survival of tropical tree seedlings enhanced by non-structural carbohydrate levels

    NASA Astrophysics Data System (ADS)

    O'Brien, Michael J.; Leuzinger, Sebastian; Philipson, Christopher D.; Tay, John; Hector, Andy

    2014-08-01

    Plants in most biomes are thought to be living at their hydraulic limits, and alterations to precipitation patterns consistent with climate change trends are causing die-back in forests across the globe. However, within- and among-species variation in plant traits that promote persistence and adaptation under these new rainfall regimes may reduce mortality in these changing climates. Storage of non-structural carbohydrates (NSCs) is posited as an important trait for resistance and resilience of forests to climate-change-induced drought, but the underlying mechanisms remain unclear. Here we demonstrate a positive relationship between NSCs and drought survival by manipulating NSC concentrations within seedlings of ten tropical tree species. Seedlings experimentally enriched in NSCs showed higher stem water potentials and sustained NSCs during drought. NSC use for maintenance of osmoregulation and hydraulic function therefore seems to underlie improved drought resistance. That drought mortality is delayed by higher NSC concentrations has implications for predicting the impacts of climate change on forest die-back and may help focus restoration efforts on species that increase the resistance and resilience of forests to climate change.

  18. Drought stress, growth and nonstructural carbohydrate dynamics of pine trees in a semi-arid forest.

    PubMed

    Klein, Tamir; Hoch, Günter; Yakir, Dan; Körner, Christian

    2014-09-01

    In trees exposed to prolonged drought, both carbon uptake (C source) and growth (C sink) typically decrease. This correlation raises two important questions: (i) to what degree is tree growth limited by C availability; and (ii) is growth limited by concurrent C storage (e.g., as nonstructural carbohydrates, NSC)? To test the relationships between drought, growth and C reserves, we monitored the changes in NSC levels and constructed stem growth chronologies of mature Pinus halepensis Miller trees of three drought stress levels growing in Yatir forest, Israel, at the dry distribution limit of forests. Moderately stressed and stressed trees showed 34 and 14% of the stem growth, 71 and 31% of the sap flux density, and 79 and 66% of the final needle length of healthy trees in 2012. In spite of these large reductions in growth and sap flow, both starch and soluble sugar concentrations in the branches of these trees were similar in all trees throughout the dry season (2-4% dry mass). At the same time, the root starch concentrations of moderately stressed and stressed trees were 47 and 58% of those of healthy trees, but never <2% dry mass. Our results show that all the studied trees maintain a fairly good coordination between C supply and demand, and even during prolonged drought there is more than one way for a tree to maintain a positive C balance.

  19. Temporal dynamics of non-structural carbohydrates and xylem growth in Pinus sylvestris exposed to drought

    PubMed Central

    Oberhuber, Walter; Swidrak, Irene; Pirkebner, Daniela; Gruber, Andreas

    2011-01-01

    Wood formation requires a continuous supply of carbohydrates for structural growth and metabolism. In the montane belt of the central Austrian Alps we monitored the temporal dynamics of xylem growth and non-structural carbohydrates (NSC) in stem sapwood of Pinus sylvestris L. during the growing season 2009, which was characterized by exceptional soil dryness within the study area. Soil water content dropped below 10 % at the time of maximum xylem growth end of May. Histological analyses have been used to describe cambial activity and xylem growth. Determination of NSC was performed using specific enzymatic assays revealing that total NSC ranged from 0.8 to 1.7 % dry matter throughout the year. Significant variations (P < 0.05) of the size of the NSC pool were observed during the growing season. Starch showed persistent abundance throughout the year reaching a maximum shortly before onset of late wood formation in mid-July. Seasonal dynamics of NSC and xylem growth suggest that (i) high sink activity occurred at start of the growing season in spring and during late wood formation in summer and (ii) there was no particular shortage in NSC, which caused P. sylvestris to draw upon stem reserves more heavily during drought in 2009. PMID:22003262

  20. Protein coding assignment of avian reovirus strain S1133.

    PubMed Central

    Varela, R; Benavente, J

    1994-01-01

    Avian reovirus S1133 encodes 10 primary translation products, 8 of which are structural components of the viral particle and 2 of which are nonstructural proteins. The identity of the gene that codes for each of these polypeptides was determined by in vitro translation of denatured individual genome segments. Images PMID:8084013

  1. Concentrations and 14C age of nonstructural carbon in California oaks

    NASA Astrophysics Data System (ADS)

    Czimczik, C. I.; Druffel-Rodriguez, K.; Trumbore, S. E.

    2008-12-01

    Plants store photosynthetic assimilates as nonstructural carbon (NSC), mainly glucose, fructose, sucrose, and starch. NSC fuels processes such as respiration and growth. Research suggests that NSC represents a significant fraction of a plant's annual C budget, but temporal dynamics of NSC are poorly understood. We used concentration and radiocarbon (14C) measurements of NSC to investigate how temporal dynamics of NSC vary with life strategy and throughout a species' range. In Mediterranean environments, oaks have developed two strategies (evergreen and deciduous) to cope with drought. Within California, the uncertainty of annual winter rain increases from north to south. We compared two evergreen and deciduous species: Coastal and Interior live oak (Quercus agrifolia and wislizenii) and Valley and Blue oak (Q. lobata and douglasii). Samples (4 mm cores to 20 cm depth at dbh) were taken in 2008 before leaf-out and fall at five sites which represent an inland to coast temperature gradient from high to low summer temperatures as well as a north- south precipitation gradient. Sugars were isolated by shaking in methanol-water and quantified using a spectrometric micro-plate technique. Starch was isolated by boiling in ethanol followed by HCl digestion and quantified manometrically. 14C contents were measured by AMS. Preliminary findings indicate that in live oaks, winter sugar concentrations are constant throughout the tree and across sites, while 14C concentrations increase towards a tree's center. This suggests that the NSC pool oaks is not well mixed. Future work will elucidate whether plants can access these older NSC stores.

  2. Molecular phylogeny and evolutionary dynamics of influenza A nonstructural (NS) gene.

    PubMed

    Xu, Jianpeng; Zhong, Haizhen A; Madrahimov, Alex; Helikar, Tomáš; Lu, Guoqing

    2014-03-01

    While the nonstructural gene (NS) of the influenza A virus plays a crucial role in viral virulence and replication, the complete understanding of its molecular phylogeny and evolutionary dynamics remains lacking. In this study, the phylogenetic analysis of 7581 NS sequences revealed ten distinct lineages within alleles A and B: three host-specific (human, classical swine and equine), two reassortment-originated (A(H1N1)pdm09 and triple reassortment swine), one transmission-originated (Eurasian swine), and two geographically isolated avian (Eurasian/Oceanian and North American) for allele A and two geographically isolated avian (Eurasian/Oceanian and North American) for allele B. The average nucleotide substitution rates of the lineages range from 1.24×10(-3) (equine) to 4.34×10(-3) (A(H1N1)pdm09) substitutions per site per year. The selection pressure analysis demonstrated that the dN/dS ratio of the NS gene in A(H1N1)pdm09 lineage was higher than its closely related triple reassortant swine, which could be attributed to the adaptation to the new host and/or intensive surveillance after the inter-species transmission from swine to human. The positive selection sites were found in all lineages except the equine lineage and mostly in the NS1 region. The positive selection sites 22, 26, 226, 227 and 230 of the human lineage are significant because these residues participate in either forming the dimerization of the two RNA binding domain (RBD) monomers or blocking the replication of host genes. Residues at position 171 provide hydrophobic interactions with hydrophobic residues at p85β and thus induce viral cell growth. The lineages and evolutionary dynamics of influenza A NS gene obtained in this study, along with the studies of other gene segments, are expected to improve the early detection of new viruses and thus have the potential to enhance influenza surveillance.

  3. Classical Swine Fever Virus p7 protein is a viroporin involved in virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The non-structural protein p7 of Classical Swine Fever Virus (CSFV) is a hydrophobic polypeptide with an apparent molecular mass of 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytos...

  4. Combined effects of defoliation and water stress on pine growth and non-structural carbohydrates.

    PubMed

    Jacquet, Jean-Sébastien; Bosc, Alexandre; O'Grady, Anthony; Jactel, Hervé

    2014-04-01

    Climate change is expected to increase both pest insect damage and the occurrence of severe drought. There is therefore a need to better understand the combined effects of biotic and abiotic damage on tree growth in order to predict the multi-factorial effect of climate change on forest ecosystem productivity. Indeed, the effect of stress interactions on tree growth is an increasingly important topic that greatly lacks experiments and data, and it is unlikely that the impact of combined stresses can be extrapolated from the outcomes of studies that focused on a single stress. We developed an original manipulative study under real field conditions where we applied artificial defoliation and induced water stress on 10-year-old (∼10 m high) maritime pine trees (Pinus pinaster Ait.). Tree response to combined stresses was quantitatively assessed following tree secondary growth and carbohydrate pools. Such a design allowed us to address the crucial question of combined stresses on trees under stand conditions, sharing soil supplies with neighboring trees. Our initial hypotheses were that (i) moderate defoliation can limit the impact of water stress on tree growth through reduced transpiration demand by a tree canopy partly defoliated and that (ii) defoliation results in reduced non-structural carbohydrate (NSC) pools, affecting tree tolerance to drought. Our results showed additive effects of defoliation and water stress on tree growth and contradict our initial hypothesis. Indeed, under stand conditions, we found that partial defoliation does not limit the impact of water stress through reduced transpiration. Our study also highlighted that, even if NSC in all organs were affected by defoliation, tree response to water stress was not triggered. We found that stem NSC were maintained or increased during the entire growing season, supporting literature-based hypotheses such as an active maintenance of the hydraulic system or another limiting resource for tree growth

  5. Mean age, concentrations and usage of nonstructural carbon in California oaks

    NASA Astrophysics Data System (ADS)

    Czimczik, C. I.; Muhr, J.; Xu, X.; Druffel-Rodriguez, K. C.; Trumbore, S.

    2012-12-01

    Recent studies show that plants transport, accumulate, and store significant amounts of photosynthetic assimilates as nonstructural carbon (NSC). However, the temporal dynamics of the NSC pool and its role as energy source, in particular during times of stress, are not well known. Taking advantage of the bomb radiocarbon (14C) tracer, we determined the mean age (here defined as mean time elapsed since C was initially fixed from the atmosphere) of soluble and insoluble NSC pools within the stem and of stem-emitted CO2 in mature, sympatric deciduous and evergreen oaks in California. In 2008, we quantified the 14C content and concentration of soluble and insoluble NSC in up to 20 cm long stem increment cores of sympatric evergreen (Quercus agrifolia, wislizeni) and deciduous (Q. lobata, douglasii) oaks during the wet (deciduous dormant) and dry (deciduous growing) seasons. Samples were taken along a coastal precipitation gradient at five nature reserves. In 2010 and 2011, we monitored the rate and 14C content of CO2 emissions from tree trunks of sympatric evergreen Q. agrifolia and deciduous Q. lobata and douglasii at two of the reserves. In all cores, we found that the NSC associated with a given depth averaged several years younger than the structural C (cellulose) in the wood at the same depth. For example, in wood made of structural C fixed before 1950, we detected modern NSC that was clearly fixed during the decades since the testing of atomic weapons in the atmosphere (peak 1950-1960s). These patterns can be explained by a model in which NSC in a given location in the stem is derived from a component that was fixed in the same year as the structural C, mixed with younger NSC that has been transported inward. Despite differences in growth rates, we found no differences in the mean age of NSC pools between life strategies or locations. Within a given stem core increment, there was no difference between the mean age of soluble and insoluble NSC. Concentrations of

  6. The influence of elevated CO2 on non-structural carbohydrate distribution and fructan accumulation in wheat canopies

    NASA Technical Reports Server (NTRS)

    Smart, D. R.; Chatterton, N. J.; Bugbee, B.

    1994-01-01

    We grew 2.4 m2 wheat canopies in a large growth chamber under high photosynthetic photon flux (1000 micromoles m-2 s-1) and using two CO2 concentrations, 360 and 1200 micromoles mol-1. Photosynthetically active radiation (400-700 nm) was attenuated slightly faster through canopies grown in 360 micromoles mol-1 than through canopies grown in 1200 micromoles mol-1, even though high-CO2 canopies attained larger leaf area indices. Tissue fractions were sampled from each 5-cm layer of the canopies. Leaf tissue sampled from the tops of canopies grown in 1200 micromoles mol-1 accumulated significantly more total non-structural carbohydrate, starch, fructan, sucrose, and glucose (p < 0.05) than for canopies grown in 360 micromoles mol-1. Non-structural carbohydrate did not significantly increase in the lower canopy layers of the elevated CO2 treatment. Elevated CO2 induced fructan synthesis in all leaf tissue fractions, but fructan formation was greatest in the uppermost leaf area. A moderate temperature reduction of 10 degrees C over 5 d increased starch, fructan and glucose levels in canopies grown in 1200 micromoles mol-1, but concentrations of sucrose and fructose decreased slightly or remained unchanged. Those results may correspond with the use of fructosyl-residues and release of glucose when sucrose is consumed in fructan synthesis.

  7. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection.

    PubMed

    Plana Duran, J; Climent, I; Sarraseca, J; Urniza, A; Cortés, E; Vela, C; Casal, J I

    1997-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tübingen isolates and between 61-64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.

  8. Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus

    PubMed Central

    Oudshoorn, Diede; van der Hoeven, Barbara; Limpens, Ronald W. A. L.; Beugeling, Corrine; Snijder, Eric J.; Bárcena, Montserrat

    2016-01-01

    ABSTRACT Infection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-β treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-β-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures. PMID:27923923

  9. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  10. Variation in the concentration and age of nonstructural carbon stored in different tree tissues

    NASA Astrophysics Data System (ADS)

    Richardson, Andrew; Carbone, Mariah; Huggett, Brett; Furze, Morgan; Czimczik, Claudia I.; Xu, Xiaomei

    2014-05-01

    Trees store nonstructural carbon (NSC), in the form of sugars and starch, in the ray parenchyma cells of woody tissues. These reserves provide a carbon buffer when demand (growth, protection, or metabolism) exceeds supply (photosynthesis). This is particularly important in the context of resilience to stress and disturbance, such as might be associated with various global change factors. However, storage allocation processes and the availability of stored reserves remain poorly understood in woody plants. To better understand how NSC reserves are distributed throughout the tree, and the degree to which NSC reserves mix across ring boundaries and tissue types, we destructively sampled two 30-year-old trees (one red oak, Quercus rubra L., and one white pine, Pinus strobus L.) growing at Harvard Forest, an oak-dominated temperate forest in the northeastern United States. We analyzed stemwood samples (divided into individual rings, bark, and phloem), coarse and fine branches, and coarse (separated into three depths) and fine roots for concentrations of total sugars and starch. For a subset of samples we used the radiocarbon (14C) "bomb spike" method to estimate the mean age of extracted sugars and starch. In oak, stemwood sugar and starch concentrations were highest (50 mg/g) in the youngest (most recently-formed) rings, and dropped off rapidly (to 10 mg/g or less) across the 10 most recent rings. In oak phloem tissue, sugar concentrations were high (90 mg/g) compared to starch (10 mg/g). In pine, sugar concentrations dropped off rapidly across the three most recent rings (from 30 mg/g to 10 mg/g) whereas starch concentrations were low even for the youngest rings (10 mg/g or less). In pine, phloem concentrations of both sugar (190 mg/g) and starch (20 mg/g) were both substantially higher than in oak. Such strong radial trends must be accounted for when scaling up to whole-tree budgets, as whole increment cores cannot properly integrate (on a ring-area basis) across the

  11. Diurnal and Seasonal Patterns of Non-Structural Carbohydrates in Pinus edulis and Juniperus monosperma

    NASA Astrophysics Data System (ADS)

    Woodruff, D.; Meinzer, F. C.; McDowell, N. G.

    2013-12-01

    Increases in drought-induced tree mortality have recently been documented globally and although less documented, non-lethal reductions in growth that are associated with the observed occurrences of tree mortality could represent an even greater drought-induced loss of net ecosystem productivity. Although carbon starvation has received a great deal of attention recently as a potential cause of drought-related mortality, the role of carbon depletion in the growth reduction and mortality of trees remains unresolved. The difference in mortality rates of piñon pine (Pinus edulis) and one seed juniper (Juniperus monosperma) in response to drought has been hypothesized to be related to their contrasting strategies for avoiding tissue desiccation and hydraulic failure (isohydry for piñon and anisohydry for juniper), and the subsequent impact of these strategies on their carbon balance. Despite intense interest in the role of carbon dynamics in tree survival and productivity, little is known of the short- and long-term consequences of drought on carbon storage and depletion in the context of these two contrasting strategies. Diurnal patterns of concentrations of non-structural carbohydrates (NSC) were analyzed in piñon and juniper under ambient growing conditions as well as under experimental drought conditions during May, August, and September of 2012. Our objective was to examine differences in storage, depletion and conversion of starch and sugars in these species at multiple points throughout the growing season and to identify any potential patterns of storage or conversion that could represent distinct vulnerabilities or compensatory responses to drought. Differences in total NSC between species were least pronounced at the beginning of the growing season in May and substantially more pronounced in August and September when NSC in piñon was reduced to approximately 2% tissue dry wt., down from 13-16% during May, whereas NSC in juniper was reduced to 4-6 % tissue

  12. Effects of structured versus non-structured learning on achievement and attitudes of fifth graders in a public aquarium

    NASA Astrophysics Data System (ADS)

    Kafka, Merryl Audrey

    The investigator analyzed the main effect of a structured-learning experience in an informal setting, as well as interactions between the students' learning-style variations toward the element of structure and the imposed instructional conditions. The subjects consisted of 170 students enrolled in two public schools located in Brooklyn, New York. The students were predominantly a White multi-ethnic population consisting of 118 Caucasians, 25 Hispanics, 24 Asians, and 3 African-Americans. Three randomly assigned classes (n = 81) were provided trip sheets, which directed students on how to learn new information with written questions and directives. Three randomly assigned non-structured classes (n = 89) experienced the same exhibit in a free-form manner. Science-based criterion-referenced pre- and posttests were administered, in addition to Learning Style Inventories (Dunn, Dunn, & Price, 1996) and a modified Semantic Differential Scale (Pizzo, 1981), which was used to measure attitudinal levels. The non-structured group had access to similar content information in the form of exhibit graphics, but apparently they chose not to read it as carefully or engage in the information-seeking process as intensely as the students equipped with trip sheets. Analysis of covariance (ANCOVA) indicated that a structured-learning experience produced significantly higher science-achievement test scores than in a non-structured-learning experience (p = .0001). In addition, there was no single learning-style variation (preference, aversion, or no preference) to structure that produced significantly higher gains than another. Furthermore, attitudinal scores were not significantly different between structured and non-structured groups, as well as among homogeneous subsets of students with learning-style variations that matched, mismatched, or indicated no-preferenced positions on the element of structure. Hence, a moderate amount of structure resulted in academic gains without

  13. Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ozone-sensitive and -tolerant individuals of the perennial herbaceous cutleaf coneflower (Rudbeckia laciniata L.) were compared for their gas exchange characteristics and total non-structural carbohydrates in the Great Smoky Mountains National Park USA. Net photosynthesis decreased with increased f...

  14. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  15. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  16. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus...

  17. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.

    PubMed

    Subissi, Lorenzo; Imbert, Isabelle; Ferron, François; Collet, Axelle; Coutard, Bruno; Decroly, Etienne; Canard, Bruno

    2014-01-01

    The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses".

  18. Identification of an NTPase motif in classical swine fever virus NS4B protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive sense single-stranded RNA virus in the genus Pestivirus of the Flaviviridae family. Here, we have identified, within CSFV non-structural (NS) protein NS4B, conserved sequence el...

  19. Proteolytic processing of Porcine Reproductive and Respiratory Syndrome Virus nsp2 replicase protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in r...

  20. The effect of replacing nonstructural carbohydrates with soybean oil on the digestibility of fibre in trotting horses.

    PubMed

    Jansen, W L; van der Kuilen, J; Geelen, S N; Beynen, A C

    2000-01-01

    The hypothesis tested was that the intake of extra fat at the expense of an isoenergetic amount of nonstructural carbohydrates reduces fibre utilisation in horses. In a crossover trial with feeding periods of 42 days each, 6 mature trotting horses (age 4-12 years, bodyweight 340-476 kg) were given either a control or test diet. The test concentrate was formulated to contain 37% of net energy in the form of soybean oil. The control concentrate contained an isoenergetic amount of corn starch plus glucose. The concentrates were fed in combination with the same amount of hay so that the control and test diet contained 25.13 and 86.66 g crude fat/kg dry matter, respectively. Apart from the amounts of fat and nonstructural carbohydrates, the 2 diets were identical. The test diet reduced the apparent total tract digestibilities of crude fibre, neutral and acid detergent fibre by 8.0 (P = 0.007), 6.2 (P = 0.022) and 8.3 (P = 0.0005) percentage units, respectively. It is suggested that a high fat intake by horses may increase the amount of fat entering the large intestine to levels that depress fermentation by cellulolytic bacteria. The observed interaction between fat content of the diet and fibre utilisation may have consequences for practical horse feeding in that calculating the energy content of test diets on the basis of feedstuff tables leads to overestimating the amount of energy provided by the high-fibre ingredients of the diets.

  1. Evaluation of Vaccines Designed to Induce Protective Cellular Immunity against the Plasmodium Yoelii Circumsporozoite Protein: Vaccinia, Pseudorabies, and Salmonella Transformed with Circumsporozoite Gene

    DTIC Science & Technology

    1990-01-01

    J, Aggarwal A, Charoenvit Y,: Hoffma’n SL 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Ya, Month, Day) jS. PAGE COUNT journ~al article ...entire P. *voei CS protein fused to then monitored for parasitaemia for 14 days. SI aminoacids of the non-structural protein of influ- hi Immunization

  2. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  3. The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

    PubMed Central

    Téoulé, François; Brisac, Cynthia; Pelletier, Isabelle; Vidalain, Pierre-Olivier; Jégouic, Sophie; Mirabelli, Carmen; Bessaud, Maël; Combelas, Nicolas; Autret, Arnaud; Tangy, Frédéric; Delpeyroux, Francis

    2013-01-01

    We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. PMID:23926333

  4. Alfalfa baleage with increased concentration of nonstructural carbohydrates supplemented with a corn-based concentrate did not improve production and nitrogen utilization in early lactation dairy cows.

    PubMed

    Brito, A F; Tremblay, G F; Bertrand, A; Castonguay, Y; Bélanger, G; Michaud, R; Lafrenière, C; Martineau, R; Berthiaume, R

    2014-11-01

    The objective of this study was to investigate the effects of feeding alfalfa baleage with different concentrations of nonstructural carbohydrates (NSC) supplemented with a common corn-based concentrate on performance, ruminal fermentation profile, N utilization, and omasal flow of nutrients in dairy cows during early lactation. Ten multiparous (8 ruminally cannulated) and 8 primiparous Holstein cows were randomly assigned to treatments (high- or low-NSC diet) in a crossover design. The difference in NSC concentration between the 2 alfalfa baleages fed from d14 to 21 averaged 14 g of NSC/kg of dry matter (DM). Forages and concentrate were offered in separate meals with forages fed once and concentrate offered 3 times daily. Except for the molar proportion of valerate, which was lowest in cows fed the high-NSC diet, no other changes in ruminal fermentation were observed. Omasal flows of most nitrogenous fractions, including bacterial nonammonia N and AA, were not affected by treatments. Apparent ruminal digestibilities of neutral and acid detergent fiber and N were lowest, whereas that of total ethanol-soluble carbohydrates was highest when feeding the high-NSC diet. Postruminal digestibilities of DM, organic matter, fiber, and N were highest in cows fed the high-NSC diet, resulting in no difference in total-tract digestibilities. Total-tract digestibility of total ethanol-soluble carbohydrates was highest in cows fed the high-NSC diet, but that of starch did not differ across treatments. Although milk yield and total DM intake did not differ between treatments, yields of milk fat and 4% fat-corrected milk decreased significantly in cows fed the high-NSC diet. Milk concentration of urea N was lowest, and that of ruminal NH3-N highest, in cows fed the high-NSC diet. Plasma urea N concentration tended to be decreased in cows fed the high-NSC diet, but concentrations of AA were not affected by treatments, with the exception of Asp and Cys, both of which were lowest in

  5. Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005

    PubMed Central

    Zohari, Siamak; Gyarmati, Péter; Ejdersund, Anneli; Berglöf, Ulla; Thorén, Peter; Ehrenberg, Maria; Czifra, György; Belák, Sándor; Waldenström, Jonas; Olsen, Björn; Berg, Mikael

    2008-01-01

    Background Although the important role of the non-structural 1 (NS) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Europe is incomplete. In this study we determined the subtypes and prevalence of influenza A viruses present in mallards in Northern Europe and further analysed the NS gene of these isolates in order to obtain a more detailed knowledge about the genetic variation of NS gene of influenza A virus in their natural hosts. Results A total number of 45 influenza A viruses of different subtypes were studied. Eleven haemagglutinin- and nine neuraminidase subtypes in twelve combinations were found among the isolated viruses. Each NS gene reported here consisted of 890 nucleotides; there were no deletions or insertions. Phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present at the same time in the same geographic location in the mallard populations in Northern Europe. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. The degree of variation within the alleles is very low. In our study allele A viruses displays a maximum of 5% amino acid divergence while allele B viruses display only 2% amino acid divergence. All the viruses isolated from mallards in Northern Europe possessed the typical avian ESEV amino acid sequence at the C-terminal end of the NS1 protein. Conclusion Our finding indicates the existence of a large reservoir of different influenza A viruses in mallards population in Northern Europe. Although our phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present in the mallards populations in Northern Europe, allele B viruses appear to be less common in natural host species

  6. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of avian influenza (AI) vaccination in poultry would have greater world-wide acceptance if a reliable test that clearly discriminates naturally infected from vaccinated only animals (DIVA) was available. Because the non-structural protein (NS1) is expressed in infected cells, and is not pac...

  7. Viral diversity and diversification of major non-structural genes vif, vpr, vpu, tat exon 1 and rev exon 1 during primary HIV-1 subtype C infection.

    PubMed

    Rossenkhan, Raabya; Novitsky, Vladimir; Sebunya, Theresa K; Musonda, Rosemary; Gashe, Berhanu A; Essex, M

    2012-01-01

    To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; -0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

  8. Linking nonstructural carbohydrate dynamics to gas exchange and leaf hydraulic behavior in Pinus edulis and Juniperus monosperma.

    PubMed

    Woodruff, David R; Meinzer, Frederick C; Marias, Danielle E; Sevanto, Sanna; Jenkins, Michael W; McDowell, Nate G

    2015-04-01

    Leaf hydraulics, gas exchange and carbon storage in Pinus edulis and Juniperus monosperma, two tree species on opposite ends of the isohydry-anisohydry spectrum, were analyzed to examine relationships between hydraulic function and carbohydrate dynamics. Leaf hydraulic vulnerability, leaf water potential (Ψl ), leaf hydraulic conductance (Kleaf ), photosynthesis (A), stomatal conductance (gs) and nonstructural carbohydrate (NSC) content were analyzed throughout the growing season. Leaf hydraulic vulnerability was significantly lower in the relatively anisohydric J. monosperma than in the more isohydric P. edulis. In P. edulis, Ψl dropped and stayed below 50% loss of leaf hydraulic conductance (P₅₀) early in the day during May, August and around midday in September, leading to sustained reductions in Kleaf . In J. monosperma, Ψl dropped below P₅₀ only during August, resulting in the maintenance of Kleaf during much of the growing season. Mean A and gs during September were significantly lower in P. edulis than in J. monosperma. Foliar total NSC was two to three times greater in J. monosperma than in P. edulis in June, August and September. Consistently lower levels of total NSC in P. edulis suggest that its isohydric strategy pushes it towards the exhaustion of carbon reserves during much of the growing season.

  9. Intra-annual dynamics of non-structural carbohydrates in the cambium of mature conifer trees reflects radial growth demands.

    PubMed

    Simard, Sonia; Giovannelli, Alessio; Treydte, Kerstin; Traversi, Maria Laura; King, Gregory M; Frank, David; Fonti, Patrick

    2013-09-01

    The presence of soluble carbohydrates in the cambial zone, either from sugars recently produced during photosynthesis or from starch remobilized from storage organs, is necessary for radial tree growth. However, considerable uncertainties on carbohydrate dynamics and the consequences on tree productivity exist. This study aims to better understand the variation in different carbon pools at intra-annual resolution by quantifying how cambial zone sugar and starch concentrations fluctuate over the season and in relation to cambial phenology. A comparison between two physiologically different species growing at the same site, i.e., the evergreen Picea abies Karst. and the deciduous Larix decidua Mill., and between L. decidua from two contrasting elevations, is presented to identify mechanisms of growth limitation. Results indicate that the annual cycle of sugar concentration within the cambial zone is coupled to the process of wood formation. The highest sugar concentration is observed when the number of cells in secondary wall formation and lignification stages is at a maximum, subsequent to most radial growth. Starch disappears in winter, while other freeze-resistant non-structural carbohydrates (NSCs) increase. Slight differences in NSC concentration between species are consistent with the differing climate sensitivity of the evergreen and deciduous species investigated. The general absence of differences between elevations suggests that the cambial activity of trees growing at the treeline was not limited by the availability of carbohydrates at the cambial zone but instead by environmental controls on the growing season duration.

  10. Leaf responses of micropropagated apple plants to water stress: nonstructural carbohydrate composition and regulatory role of metabolic enzymes.

    PubMed

    Li, Tian H; Li, Shao H

    2005-04-01

    We examined changes in nonstructural carbohydrate biosynthesis and activities of related enzymes in leaves of micropropagated apple plants (Malus domestica Borkh. cv. 'NaganoFuji') in response to water stress, with particular emphasis on the enzymes associated with sorbitol, sucrose and starch metabolism. Water stress resulted in the accumulation of photosynthates in leaves, mainly sorbitol, sucrose, glucose and fructose, accompanied by a reduction in starch concentration. Correlation and path analysis indicated that water stress affected the partitioning of newly fixed carbon among terminal products. In response to water stress, ADP-glucose-pyrophosphorylase (ADPGPPase) activity decreased, becoming a critical and limiting step in shifting partitioning of photosynthetically fixed carbon. Amylase and ADPGPPase affected sucrose and sorbitol metabolism, mainly by regulating substrate supply; however, competition for limited substrate had a greater effect on the biosynthesis of sorbitol than of sucrose. Starch metabolism was also strictly regulated by ADPGPPase and amylase, whereas other related enzymes were downstream of the pathway for synthesis and degradation of carbohydrates and thus had relatively little effect on starch metabolism. Sorbitol dehydrogenase and sucrose phosphate synthase were critical regulators of sorbitol and sucrose metabolism, respectively.

  11. Non-structural carbohydrate partitioning in grass stems: a target to increase yield stability, stress tolerance, and biofuel production.

    PubMed

    Slewinski, Thomas L

    2012-08-01

    A dramatic change in agricultural crops is needed in order to keep pace with the demands of an increasing human population, exponential need for renewable fuels, and uncertain climatic changes. Grasses make up the vast majority of agricultural commodities. How these grasses capture, transport, and store carbohydrates underpins all aspects of crop productivity. Sink-source dynamics within the plant direct how much, where, and when carbohydrates are allocated, as well as determine the harvestable tissue. Carbohydrate partitioning can limit the yield capacity of these plants, thus offering a potential target for crop improvement. Grasses have the ability to buffer this sink-source interaction by transiently storing carbohydrates in stem tissue when production from the source is greater than whole-plant demand. These reserves improve yield stability in grain crops by providing an alternative source when photosynthetic capacity is reduced during the later phases of grain filling, or during periods of environmental and biotic stresses. Domesticated grasses such as sugarcane and sweet sorghum have undergone selection for high accumulation of stem carbohydrates, which serve as the primary sources of sugars for human and animal consumption, as well as ethanol production for fuel. With the enormous expectations placed on agricultural production in the near future, research into carbohydrate partitioning in grasses is essential for maintaining and increasing yields in grass crops. This review highlights the current knowledge of non-structural carbohydrate dynamics in grass stems and discusses the impacts of stem reserves in essential agronomic grasses.

  12. Sweets for the foe - effects of nonstructural carbohydrates on the susceptibility of Quercus robur against Phytophthora quercina.

    PubMed

    Angay, Oguzhan; Fleischmann, Frank; Recht, Sabine; Herrmann, Sylvie; Matyssek, Rainer; Oßwald, Wolfgang; Buscot, François; Grams, Thorsten E E

    2014-09-01

    The root-rot pathogen Phytophthora quercina is a key determinant of oak decline in Europe. The susceptibility of pedunculate oak (Quercus robur) to this pathogen has been hypothesized to depend on the carbon availability in roots as an essential resource for defense. Microcuttings of Q. robur undergo an alternating rhythm of root and shoot growth. Inoculation of mycorrhizal (Piloderma croceum) and nonmycorrhizal oak roots with P. quercina was performed during both growth phases, that is, root flush (RF) and shoot flush (SF). Photosynthetic and morphological responses as well as concentrations of nonstructural carbohydrates (NSC) were analyzed. Infection success was quantified by the presence of pathogen DNA in roots. Concentrations of NSC in roots depended on the alternating root/shoot growth rhythm, being high and low during RF and SF, respectively. Infection success was high during RF and low during SF, resulting in a significantly positive correlation between pathogen DNA and NSC concentration in roots, contrary to the hypothesis. The alternating growth of roots and shoots plays a crucial role for the susceptibility of lateral roots to the pathogen. NSC availability in oak roots has to be considered as a benchmark for susceptibility rather than resistance against P. quercina.

  13. Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

    PubMed

    Babst, Benjamin A; Karve, Abhijit A; Judt, Tatjana

    2013-06-01

    Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

  14. Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

    PubMed Central

    Rothman, A L; Kurane, I; Lai, C J; Bray, M; Falgout, B; Men, R; Ennis, F A

    1993-01-01

    The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3. CTL from H-2b mice recognized a serotype-cross-reactive epitope that was localized to NS4a or NS4b. CTL from H-2d mice recognized at least three epitopes: a serotype-specific epitope on one of the structural proteins, a serotype-cross-reactive epitope on NS3, and a serotype-cross-reactive epitope on NS1 or NS2a. Our findings demonstrate the limited recognition of dengue virus proteins by CTL from three inbred mouse strains and the predominance of CTL epitopes on dengue virus nonstructural proteins, particularly NS3. Since human dengue virus-specific CTL show similar patterns of recognition, these findings suggest that nonstructural proteins should be considered in designing vaccines against dengue. PMID:7678307

  15. Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

    PubMed Central

    Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K.; Nichol, Stuart T.; Albariño, César G.; Spiropoulou, Christina F.

    2015-01-01

    Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public

  16. Feedbacks between earlywood anatomy and non-structural carbohydrates affect spring phenology and wood production in ring-porous oaks

    NASA Astrophysics Data System (ADS)

    Pérez-de-Lis, Gonzalo; García-González, Ignacio; Rozas, Vicente; Olano, José Miguel

    2016-10-01

    Non-structural carbohydrates (NSC) play a central role in the construction and maintenance of a tree's vascular system, but feedbacks between the NSC status of trees and wood formation are not fully understood. We aimed to evaluate multiple dependencies among wood anatomy, winter NSC, and phenology for coexisting temperate (Quercus robur) and sub-Mediterranean (Q. pyrenaica) oaks along a water-availability gradient in the NW Iberian Peninsula. Sapwood NSC concentrations were quantified at three sites in December 2012 (N = 240). Leaf phenology and wood anatomy were surveyed in 2013. Structural equation modelling was used to analyse the interplay among hydraulic diameter (Dh), winter NSC, budburst date, and earlywood vessel production (EVP), while the effect of Dh and EVP on latewood width was assessed by using a mixed-effects model. NSC and wood production increased under drier conditions for both species. Q. robur showed a narrower Dh and lower soluble sugar (SS) concentration (3.88-5.08 % dry matter) than Q. pyrenaica (4.06-5.57 % dry matter), but Q. robur exhibited larger EVP and wider latewood (1403 µm) than Q. pyrenaica (667 µm). Stem diameter and Dh had a positive effect on SS concentrations, which were related to an earlier leaf flushing in both species. Sapwood sugar content appeared to limit EVP exclusively in Q. pyrenaica. In turn, Dh and EVP were found to be key predictors of latewood growth. Our results confirm that sapwood SS concentrations are involved in modulating growth resumption and xylem production in spring. Q. pyrenaica exhibited a tighter control of carbohydrate allocation to wood formation than Q. robur, which would play a role in protecting against environmental stress in the sub-Mediterranean area.

  17. Dynamics of non-structural carbohydrates in three Mediterranean woody species following long-term experimental drought

    PubMed Central

    Rosas, Teresa; Galiano, Lucía; Ogaya, Romà; Peñuelas, Josep; Martínez-Vilalta, Jordi

    2013-01-01

    Stored non-structural carbohydrates (NSC) have been proposed as a key determinant of drought resistance in plants. However, the evidence for this role is controversial, as it comes mostly from observational, short-term studies. Here, we take advantage of a long-term experimental throughfall reduction to elucidate the response of NSC to increased drought 14 years after the beginning of the treatment in three Mediterranean resprouter trees (Quercus ilex L., Arbutus unedo L. and Phillyrea latifolia L.). In addition, we selected 20 Q. ilex individuals outside the experimental plots to directly assess the relationship between defoliation and NSC at the individual level. We measured the seasonal course of NSC concentrations in leaves, branches and lignotuber in late winter, late spring, summer, and autumn 2012. Total concentrations of NSC were highest in the lignotuber for all species. In the long-term drought experiment we found significant depletion in concentrations of total NSC in treatment plots only in the lignotuber of A. unedo. At the same time, A. unedo was the only species showing a significant reduction in BAI under the drought treatment during the 14 years of the experiment. By contrast, Q. ilex just reduced stem growth only during the first 4 years of treatment and P. latifolia remained unaffected over the whole study period. However, we found a clear association between the concentrations of NSC and defoliation in Q. ilex individuals sampled outside the experimental plots, with lower total concentrations of NSC and lower proportion of starch in defoliated individuals. Taken together, our results suggest that stabilizing processes, probably at the stand level, may have been operating in the long-term to mitigate any impact of drought on NSC levels, and highlight the necessity to incorporate long-term experimental studies of plant responses to drought. PMID:24130568

  18. Non-structural carbon dynamics and allocation relate to growth rate and leaf habit in California oaks.

    PubMed

    Trumbore, Susan; Czimczik, Claudia I; Sierra, Carlos A; Muhr, Jan; Xu, Xiaomei

    2015-11-01

    Trees contain non-structural carbon (NSC), but it is unclear for how long these reserves are stored and to what degree they are used to support plant activity. We used radiocarbon ((14)C) to show that the carbon (C) in stemwood NSC can achieve ages of several decades in California oaks. We separated NSC into two fractions: soluble (∼50% sugars) and insoluble (mostly starch) NSC. Soluble NSC contained more C than insoluble NSC, but we found no consistent trend in the amount of either pool with depth in the stem. There was no systematic difference in C age between the two fractions, although ages increased with stem depth. The C in both NSC fractions was consistently younger than the structural C from which they were extracted. Together, these results indicate considerable inward mixing of NSC within the stem and rapid exchange between soluble and insoluble pools, compared with the timescale of inward mixing. We observed similar patterns in sympatric evergreen and deciduous oaks and the largest differences among tree stems with different growth rates. The (14)C signature of carbon dioxide (CO2) emitted from tree stems was higher than expected from very recent photoassimilates, indicating that the mean age of C in respiration substrates included a contribution from C fixed years previously. A simple model that tracks NSC produced each year, followed by loss (through conversion to CO2) in subsequent years, matches our observations of inward mixing of NSC in the stem and higher (14)C signature of stem CO2 efflux. Together, these data support the idea of continuous accumulation of NSC in stemwood and that 'vigor' (growth rate) and leaf habit (deciduous vs evergreen) control NSC pool size and allocation.

  19. A synthetic codon-optimized hepatitis C virus nonstructural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wild-type and NS5A-transgenic mice.

    PubMed

    Holmström, Fredrik; Pasetto, Anna; Nähr, Veronica; Brass, Anette; Kriegs, Malte; Hildt, Eberhard; Broderick, Kate E; Chen, Margaret; Ahlén, Gustaf; Frelin, Lars

    2013-02-01

    The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >10(4) postimmunization. With respect to CD8(+) T cell responses, the coNS5A gene primed more potent IFN-γ-producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8(+) T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8(+) T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

  20. Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

    PubMed

    Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V; Walton, Ross; Johnston, Sebastian L; Solari, Roberto

    2014-10-01

    The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be

  1. Nucleocytoplasmic shuttling of influenza A virus proteins.

    PubMed

    Li, Jing; Yu, Meng; Zheng, Weinan; Liu, Wenjun

    2015-05-22

    Influenza viruses transcribe and replicate their genomes in the nuclei of infected host cells. The viral ribonucleoprotein (vRNP) complex of influenza virus is the essential genetic unit of the virus. The viral proteins play important roles in multiple processes, including virus structural maintenance, mediating nucleocytoplasmic shuttling of the vRNP complex, virus particle assembly, and budding. Nucleocytoplasmic shuttling of viral proteins occurs throughout the entire virus life cycle. This review mainly focuses on matrix protein (M1), nucleoprotein (NP), nonstructural protein (NS1), and nuclear export protein (NEP), summarizing the mechanisms of their nucleocytoplasmic shuttling and the regulation of virus replication through their phosphorylation to further understand the regulation of nucleocytoplasmic shuttling in host adaptation of the viruses.

  2. Osmolality and Non-Structural Carbohydrate Composition in the Secondary Phloem of Trees across a Latitudinal Gradient in Europe

    PubMed Central

    Lintunen, Anna; Paljakka, Teemu; Jyske, Tuula; Peltoniemi, Mikko; Sterck, Frank; von Arx, Georg; Cochard, Hervé; Copini, Paul; Caldeira, Maria C.; Delzon, Sylvain; Gebauer, Roman; Grönlund, Leila; Kiorapostolou, Natasa; Lechthaler, Silvia; Lobo-do-Vale, Raquel; Peters, Richard L.; Petit, Giai; Prendin, Angela L.; Salmon, Yann; Steppe, Kathy; Urban, Josef; Roig Juan, Sílvia; Robert, Elisabeth M. R.; Hölttä, Teemu

    2016-01-01

    Phloem osmolality and its components are involved in basic cell metabolism, cell growth, and in various physiological processes including the ability of living cells to withstand drought and frost. Osmolality and sugar composition responses to environmental stresses have been extensively studied for leaves, but less for the secondary phloem of plant stems and branches. Leaf osmotic concentration and the share of pinitol and raffinose among soluble sugars increase with increasing drought or cold stress, and osmotic concentration is adjusted with osmoregulation. We hypothesize that similar responses occur in the secondary phloem of branches. We collected living bark samples from branches of adult Pinus sylvestris, Picea abies, Betula pendula and Populus tremula trees across Europe, from boreal Northern Finland to Mediterranean Portugal. In all studied species, the observed variation in phloem osmolality was mainly driven by variation in phloem water content, while tissue solute content was rather constant across regions. Osmoregulation, in which osmolality is controlled by variable tissue solute content, was stronger for Betula and Populus in comparison to the evergreen conifers. Osmolality was lowest in mid-latitude region, and from there increased by 37% toward northern Europe and 38% toward southern Europe due to low phloem water content in these regions. The ratio of raffinose to all soluble sugars was negligible at mid-latitudes and increased toward north and south, reflecting its role in cold and drought tolerance. For pinitol, another sugar known for contributing to stress tolerance, no such latitudinal pattern was observed. The proportion of sucrose was remarkably low and that of hexoses (i.e., glucose and fructose) high at mid-latitudes. The ratio of starch to all non-structural carbohydrates increased toward the northern latitudes in agreement with the build-up of osmotically inactive C reservoir that can be converted into soluble sugars during winter

  3. Analysis of nonstructural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection.

    PubMed

    Ranwala, Anil P; Miller, William B

    2008-01-01

    A comprehensive analysis of nonstructural carbohydrates in storage organs (bulbs and corms) of 30 ornamental geophytes was conducted by employing a variety of extraction techniques followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Among species, starch, fructan, glucomannan and soluble sugars accounted for 50-80% of storage organ dry weight (DW). Starch ranged from 24 to 760 mg g(-1) DW, fructan (commonly occurring with starch) from 25 to 500 mg g(-1) DW, and glucomannan from 15 to 145 mg g(-1) DW. An acid hydrolysis protocol for concurrent determination of fructan and glucomannan was developed. The average degree of polymerization (DP) of ethanol and water-soluble fructan and the man : glu ratio of glucomannan also varied between species. The 80% ethanol fraction contained soluble sugars and short-chain fructans (< 25 DP), whereas water extracts contained soluble sugars, fructans (both short- and long-chain, nonstructural carbohydrates for physiological and biochemical research on geophytic storage organs should be validated to accurately understand the identity of diverse carbohydrate pools, their physiological relevance and functions.

  4. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue.

    PubMed

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-11-15

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

  5. Monoclonal antibodies specific for African swine fever virus proteins.

    PubMed Central

    Sanz, A; García-Barreno, B; Nogal, M L; Viñuela, E; Enjuanes, L

    1985-01-01

    We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles. Images PMID:3882998

  6. The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

    PubMed

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-02-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

  7. The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

    PubMed Central

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

    2012-01-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

  8. Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea.

    PubMed

    Reddy, Kondreddy Eswar; Noh, Jin Hyeong; Kim, Young-Ha; Yoo, Mi Sun; Doan, Huong Thi Thanh; Ramya, Mummadireddy; Jung, Suk-Chan; Quyen, Dong Van; Kang, Seung-Won

    2013-09-01

    Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

  9. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

    PubMed Central

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S.; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-01-01

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease. PMID:27845418

  10. Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast

    PubMed Central

    Li, Ge; Poulsen, Melissa; Fenyvuesvolgyi, Csaba; Yashiroda, Yoko; Yoshida, Minoru; Simard, J. Marc; Gallo, Robert C.; Zhao, Richard Y.

    2017-01-01

    The Zika virus (ZIKV) causes microcephaly and the Guillain-Barré syndrome. Little is known about how ZIKV causes these conditions or which ZIKV viral protein(s) is responsible for the associated ZIKV-induced cytopathic effects, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. We used fission yeast for the rapid, global functional analysis of the ZIKV genome. All 14 proteins or small peptides were produced under an inducible promoter, and we measured the intracellular localization and the specific effects on ZIKV-associated cytopathic activities of each protein. The subcellular localization of each ZIKV protein was in overall agreement with its predicted protein structure. Five structural and two nonstructural ZIKV proteins showed various levels of cytopathic effects. The expression of these ZIKV proteins restricted cell proliferation, induced hypertrophy, or triggered cellular oxidative stress leading to cell death. The expression of premembrane protein (prM) resulted in cell-cycle G1 accumulation, whereas membrane-anchored capsid (anaC), membrane protein (M), envelope protein (E), and nonstructural protein 4A (NS4A) caused cell-cycle G2/M accumulation. A mechanistic study revealed that NS4A-induced cellular hypertrophy and growth restriction were mediated specifically through the target of rapamycin (TOR) cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41. These findings should provide a reference for future research on the prevention and treatment of ZIKV diseases. PMID:28049830

  11. Hepatitis C Virus Co-Opts Ras-GTPase-Activating Protein-Binding Protein 1 for Its Genome Replication ▿

    PubMed Central

    Yi, Zhigang; Pan, Tingting; Wu, Xianfang; Song, Wuhui; Wang, Shanshan; Xu, Yan; Rice, Charles M.; MacDonald, Margaret R.; Yuan, Zhenghong

    2011-01-01

    We recently reported that Ras-GTPase-activating protein-binding protein 1 (G3BP1) interacts with hepatitis C virus (HCV) nonstructural protein (NS)5B and the 5′ end of the HCV minus-strand RNA. In the current study we confirmed these observations using immunoprecipitation and RNA pulldown assays, suggesting that G3BP1 might be an HCV replication complex (RC) component. In replicon cells, transfected G3BP1 interacts with multiple HCV nonstructural proteins. Using immunostaining and confocal microscopy, we demonstrate that G3BP1 is colocalized with HCV RCs in replicon cells. Small interfering RNA (siRNA)-mediated knockdown of G3BP1 moderately reduces established HCV RNA replication in HCV replicon cells and dramatically reduces HCV replication-dependent colony formation and cell-culture-produced HCV (HCVcc) infection. In contrast, knockdown of G3BP2 has no effect on HCVcc infection. Transient replication experiments show that G3BP1 is involved in HCV genome amplification. Thus, G3BP1 is associated with HCV RCs and may be co-opted as a functional RC component for viral replication. These findings may facilitate understanding of the molecular mechanisms of HCV genome replication. PMID:21561913

  12. The Rift Valley Fever virus protein NSm and putative cellular protein interactions.

    PubMed

    Engdahl, Cecilia; Näslund, Jonas; Lindgren, Lena; Ahlm, Clas; Bucht, Göran

    2012-07-28

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection.

  13. Serotype-specific interactions among functional domains of Dengue virus 2 non-structural proteins (NS) 5 and NS3 are crucial for viral RNA replication.

    PubMed

    Teramoto, Tadahisa; Balasubramanian, Anuradha; Choi, Kyung H; Padmanabhan, Radhakrishnan

    2017-04-10

    Four serotypes of mosquito-borne Dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance, and there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase) and the C-terminal region has the polymerase (POL) which are important for 5'-capping and RNA replication. The crystal structure of NS5 showed it as a dimer but the functional evidence for NS5 dimer is lacking. The results of our studies show that the substitution of DENV2 NS5 MTase or POL with that of DENV4 NS5 within DENV2 RNA resulted in severe attenuation of replication in the transfected BHK-21 cells. A replication competent species evolved with acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for serotype-specific interaction between NS3 and NS5 as well as specific inter-domain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface providing a functional significance to the crystal structure NS5 dimer.

  14. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  15. Poliovirus protein 2BC increases cytosolic free calcium concentrations.

    PubMed Central

    Aldabe, R; Irurzun, A; Carrasco, L

    1997-01-01

    Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis. PMID:9223520

  16. Rooting depth, water relations and non-structural carbohydrate dynamics in three woody angiosperms differentially affected by an extreme summer drought.

    PubMed

    Nardini, Andrea; Casolo, Valentino; Dal Borgo, Anna; Savi, Tadeja; Stenni, Barbara; Bertoncin, Paolo; Zini, Luca; McDowell, Nathan G

    2016-03-01

    In 2012, an extreme summer drought induced species-specific die-back in woody species in Northeastern Italy. Quercus pubescens and Ostrya carpinifolia were heavily impacted, while Prunus mahaleb was largely unaffected. By comparing seasonal changes in isotopic composition of xylem sap, rainfall and deep soil samples, we show that P. mahaleb has a deeper root system than the other two species. This morphological trait allowed P  mahaleb to maintain higher water potential (Ψ), gas exchange rates and non-structural carbohydrates content (NSC) throughout the summer, when compared with the other species. More favourable water and carbon states allowed relatively stable maintenance of stem hydraulic conductivity (k) throughout the growing season. In contrast, in Quercus pubescens and Ostrya carpinifolia, decreasing Ψ and NSC were associated with significant hydraulic failure, with spring-to-summer k loss averaging 60%. Our data support the hypothesis that drought-induced tree decline is a complex phenomenon that cannot be modelled on the basis of single predictors of tree status like hydraulic efficiency, vulnerability and carbohydrate content. Our data highlight the role of rooting depth in seasonal progression of water status, gas exchange and NSC, with possible consequences for energy-demanding mechanisms involved in the maintenance of vascular integrity.

  17. Dark respiration rate increases with plant size in saplings of three temperate tree species despite decreasing tissue nitrogen and nonstructural carbohydrates.

    PubMed

    Machado, José-Luis; Reich, Peter B

    2006-07-01

    In shaded environments, minimizing dark respiration during growth could be an important aspect of maintaining a positive whole-plant net carbon balance. Changes with plant size in both biomass distribution to different tissue types and mass-specific respiration rates (R(d)) of those tissues would have an impact on whole-plant respiration. In this paper, we evaluated size-related variation in R(d), biomass distribution, and nitrogen (N) and total nonstructural carbohydrate (TNC) concentrations of leaves, stems and roots of three cold-temperate tree species (Abies balsamea (L.) Mill, Acer rubrum L. and Pinus strobus L.) in a forest understory. We sampled individuals varying in age (6 to 24 years old) and in size (from 2 to 500 g dry mass), and growing across a range of irradiances (from 1 to 13% of full sun) in northern Minnesota, USA. Within each species, we found small changes in R(d), N and TNC when comparing plants growing across this range of light availability. Consistent with our hypotheses, as plants grew larger, whole-plant N and TNC concentrations in all species declined as a result of a combination of changes in tissue N and shifts in biomass distribution patterns. However, contrary to our hypotheses, whole-plant and tissue R(d) increased with plant size in the three species.

  18. Effects of high and moderate non-structural carbohydrate hay on insulin, glucose, triglyceride, and leptin concentrations in overweight Arabian geldings.

    PubMed

    Shepherd, M L; Pleasant, R S; Crisman, M V; Werre, S R; Milton, S C; Swecker, W S

    2012-06-01

    The objective of this study was to determine the effects of high and moderate non-structural carbohydrates (NSC) hay on insulin, glucose, triglyceride, and leptin concentrations in overweight Arabian geldings. Eight adult overweight (average BCS 7 [9-point scale]) Arabian geldings were fed each of two orchardgrass hays, high NSC (18% DM) and moderate NSC (12% DM), in a cross over design during two 28-day periods. Body weight and body condition score assessment along with blood sampling to measure insulin, glucose, leptin, and triglyceride concentrations were performed on days 0, 7, 14, 21 and 28 of each period. Effects of hay, period, day, and day*hay on plasma glucose and serum leptin were not detected. Serum insulin was influenced by hay (p = 0.001), day (p = 0.03), and day*hay (p = 0.04). Insulin concentrations were higher on day 7 in the high NSC group (15.6 μIU/ml) than the moderate NSC group (9.5 μIU/ml), but not by day 14 (p = 0.0007). Plasma triglyceride was influenced by period (p = 0.0003), day*period (p < 0.0001), and day*hay (p = 0.02). Hyperinsulinaemia was not observed in the overweight Arabian geldings fed either a moderate or high NSC hay.

  19. Determination of water-extractable nonstructural carbohydrates, including inulin, in grass samples with high-performance anion exchange chromatography and pulsed amperometric detection.

    PubMed

    Raessler, Michael; Wissuwa, Bianka; Breul, Alexander; Unger, Wolfgang; Grimm, Torsten

    2008-09-10

    The exact and reliable determination of carbohydrates in plant samples of different origin is of great importance with respect to plant physiology. Additionally, the identification and quantification of carbohydrates are necessary for the evaluation of the impact of these compounds on the biogeochemistry of carbon. To attain this goal, it is necessary to analyze a great number of samples with both high sensitivity and selectivity within a limited time frame. This paper presents a rugged and easy method that allows the isocratic chromatographic determination of 12 carbohydrates and sugar alcohols from one sample within 30 min. The method was successfully applied to a variety of plant materials with particular emphasis on perennial ryegrass samples of the species Lolium perenne. The method was easily extended to the analysis of the polysaccharide inulin after its acidic hydrolysis into the corresponding monomers without the need for substantial change of chromatographic conditions or even the use of enzymes. It therefore offers a fundamental advantage for the analysis of the complex mixture of nonstructural carbohydrates often found in plant samples.

  20. Genotypic variations in non-structural carbohydrate and cell-wall components of the stem in rice, sorghum, and sugar vane.

    PubMed

    Arai-Sanoh, Yumiko; Ida, Masashi; Zhao, Rui; Yoshinaga, Satoshi; Takai, Toshiyuki; Ishimaru, Tsutomu; Maeda, Hideo; Nishitani, Kazuhiko; Terashima, Yoshifumi; Gau, Mitsuru; Kato, Naoki; Matsuoka, Makoto; Kondo, Motohiko

    2011-01-01

    We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.

  1. Correlation of the 5'untranslated region (5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping.

    PubMed

    Baclig, Michael O; Chan, Veronica F; Ramos, John Donnie A; Gopez-Cervantes, Juliet; Natividad, Filipinas F

    2010-07-07

    The 5'untranslated region (5'UTR) is often targeted to detect major genotypes in hepatitis C virus (HCV) but its insufficient sequence variation limits its usefulness for differentiating HCV subtypes. Subtyping has important implications to epidemiologic studies, clinical management, and vaccine development. Analysis of the nucleotide sequence of variable regions such as the non-structural 5B (NS5B) is considered the reference method for identifying HCV subtypes. We evaluated the accuracy of subtyping of HCV genotype 1 (HCV-1) samples from the Philippines by 5'UTR sequencing as compared with the NS5B sequence. A total of 30 patients infected with HCV-1 previously confirmed by PCR-RFLP and clinically diagnosed with chronic hepatitis C were analyzed. Nucleotide sequencing of the 5'UTR showed that 15 (50%) were identified as 1a and 15 (50%) were identified as 1b. Sequence analysis of the NS5B revealed that 13 (43%) belonged to subtype 1a while 17 (57%) belonged to subtype 1b. The most predominant subtype was 1b by NS5B sequencing. The predictive value of 5'UTR sequencing to subtype 1a was 73% while for subtype 1b, predictive value was 87%. Overall concordance between 5'UTR and NS5B sequencing was 80%. NS5B sequence and phylogenetic analysis is still the reference method for identifying HCV-1a and 1b subtypes.

  2. Correlation of the 5′untranslated region (5′UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping

    PubMed Central

    Baclig, Michael O; Chan, Veronica F; Ramos, John Donnie A; Gopez-Cervantes, Juliet; Natividad, Filipinas F

    2010-01-01

    The 5′untranslated region (5′UTR) is often targeted to detect major genotypes in hepatitis C virus (HCV) but its insufficient sequence variation limits its usefulness for differentiating HCV subtypes. Subtyping has important implications to epidemiologic studies, clinical management, and vaccine development. Analysis of the nucleotide sequence of variable regions such as the non-structural 5B (NS5B) is considered the reference method for identifying HCV subtypes. We evaluated the accuracy of subtyping of HCV genotype 1 (HCV-1) samples from the Philippines by 5′UTR sequencing as compared with the NS5B sequence. A total of 30 patients infected with HCV-1 previously confirmed by PCR-RFLP and clinically diagnosed with chronic hepatitis C were analyzed. Nucleotide sequencing of the 5′UTR showed that 15 (50%) were identified as 1a and 15 (50%) were identified as 1b. Sequence analysis of the NS5B revealed that 13 (43%) belonged to subtype 1a while 17 (57%) belonged to subtype 1b. The most predominant subtype was 1b by NS5B sequencing. The predictive value of 5′UTR sequencing to subtype 1a was 73% while for subtype 1b, predictive value was 87%. Overall concordance between 5′UTR and NS5B sequencing was 80%. NS5B sequence and phylogenetic analysis is still the reference method for identifying HCV-1a and 1b subtypes. PMID:21537395

  3. Dynamic allocation and transfer of non-structural carbohydrates, a possible mechanism for the explosive growth of Moso bamboo (Phyllostachys heterocycla)

    PubMed Central

    Song, Xinzhang; Peng, Changhui; Zhou, Guomo; Gu, Honghao; Li, Quan; Zhang, Chao

    2016-01-01

    Moso bamboo can rapidly complete its growth in both height and diameter within only 35–40 days after shoot emergence. However, the underlying mechanism for this “explosive growth” remains poorly understood. We investigated the dynamics of non-structural carbohydrates (NSCs) in shoots and attached mature bamboos over a 20-month period. The results showed that Moso bamboos rapidly completed their height and diameter growth within 38 days. At the same time, attached mature bamboos transferred almost all the NSCs of their leaves, branches, and especially trunks and rhizomes to the “explosively growing” shoots via underground rhizomes for the structural growth and metabolism of shoots. Approximately 4 months after shoot emergence, this transfer stopped when the leaves of the young bamboos could independently provide enough photoassimilates to meet the carbon demands of the young bamboos. During this period, the NSC content of the leaves, branches, trunks and rhizomes of mature bamboos declined by 1.5, 23, 28 and 5 fold, respectively. The trunk contributed the most NSCs to the shoots. Our findings provide new insight and a possible rational mechanism explaining the “explosive growth” of Moso bamboo and shed new light on understanding the role of NSCs in the rapid growth of Moso bamboo. PMID:27181522

  4. Dynamic allocation and transfer of non-structural carbohydrates, a possible mechanism for the explosive growth of Moso bamboo (Phyllostachys heterocycla)

    NASA Astrophysics Data System (ADS)

    Song, Xinzhang; Peng, Changhui; Zhou, Guomo; Gu, Honghao; Li, Quan; Zhang, Chao

    2016-05-01

    Moso bamboo can rapidly complete its growth in both height and diameter within only 35–40 days after shoot emergence. However, the underlying mechanism for this “explosive growth” remains poorly understood. We investigated the dynamics of non-structural carbohydrates (NSCs) in shoots and attached mature bamboos over a 20-month period. The results showed that Moso bamboos rapidly completed their height and diameter growth within 38 days. At the same time, attached mature bamboos transferred almost all the NSCs of their leaves, branches, and especially trunks and rhizomes to the “explosively growing” shoots via underground rhizomes for the structural growth and metabolism of shoots. Approximately 4 months after shoot emergence, this transfer stopped when the leaves of the young bamboos could independently provide enough photoassimilates to meet the carbon demands of the young bamboos. During this period, the NSC content of the leaves, branches, trunks and rhizomes of mature bamboos declined by 1.5, 23, 28 and 5 fold, respectively. The trunk contributed the most NSCs to the shoots. Our findings provide new insight and a possible rational mechanism explaining the “explosive growth” of Moso bamboo and shed new light on understanding the role of NSCs in the rapid growth of Moso bamboo.

  5. Effects of irradiance on non-structural carbohydrates, growth, and hypoglycemic activity of Rhynchelytrum repens (Willd.) C.E. Hubb. (Poaceae).

    PubMed

    Souza, A; de Paula, A C C F F; Figueiredo-Ribeiro, R C L

    2004-08-01

    Irradiance is a crucial factor in plant development. Different radiant energy levels cause different responses related to plant growth and the production and distribution of dry matter. Considering the relationship between light availability and metabolism of carbohydrates and derived compounds, the aim of this work was to assess the effects of irradiance on non-structural carbohydrate contents and composition, as well as on the hypoglycemiant activity of Rhynchelytrum repens, a pantropical grass species popularly used for diabetes treatment. Plants of R. repens growing under natural irradiance (NI) showed increased content of total soluble carbohydrate (TSC), higher fluctuations in starch content (SC) and higher number of tillers. The flowering process of these plants was preceded by an increase in sucrose. However, their water content was low when compared to that of plants cultivated under low irradiance (LI). The ratio root/aerial organs and SC showed no significant differences in plants grown under LI, although TSC increased and a lower number of tillers were observed during the experimental period. In both conditions, sucrose was the ubiquitous sugar and seemed to be involved in the flowering process. A reduction in the blood sugar level was observed through the intra-peritoneal (IP) administration of the precipitate of aqueous extracts obtained from plants growing in both conditions of light; the supernatant fraction showed no hypoglycemic effect.

  6. Drought-induced shoot dieback starts with massive root xylem embolism and variable depletion of nonstructural carbohydrates in seedlings of two tree species.

    PubMed

    Rodríguez-Calcerrada, Jesús; Li, Meng; López, Rosana; Cano, Francisco Javier; Oleksyn, Jacek; Atkin, Owen K; Pita, Pilar; Aranda, Ismael; Gil, Luis

    2017-01-01

    Combining hydraulic- and carbon-related measurements helps to understand drought-induced plant mortality. Here, we investigated the role that plant respiration (R) plays in determining carbon budgets under drought. We measured the hydraulic conductivity of stems and roots, and gas exchange and nonstructural carbohydrate (NSC) concentrations of leaves, stems and roots of seedlings of two resprouting species exposed to drought or well-watered conditions: Ulmus minor (riparian tree) and Quercus ilex (dryland tree). With increasing water stress (occurring more rapidly in larger U. minor), declines in leaf, stem and root R were less pronounced than that in leaf net photosynthetic CO2 uptake (Pn ). Daytime whole-plant carbon gain was negative below -4 and -6 MPa midday xylem water potential in U. minor and Q. ilex, respectively. Relative to controls, seedlings exhibiting shoot dieback suffered c. 80% loss of hydraulic conductivity in both species, and reductions in NSC concentrations in U. minor. Higher drought-induced depletion of NSC reserves in U. minor was related to higher plant R, faster stomatal closure, and premature leaf-shedding. Differences in drought resistance relied on the ability to maintain hydraulic conductivity during drought, rather than tolerating conductivity loss. Root hydraulic failure elicited shoot dieback and precluded resprouting without root NSC reserves being apparently limiting for R.

  7. Ectomycorrhizal identity determines respiration and concentrations of nitrogen and non-structural carbohydrates in root tips: a test using Pinus sylvestris and Quercus robur saplings.

    PubMed

    Trocha, Lidia K; Mucha, Joanna; Eissenstat, David M; Reich, Peter B; Oleksyn, Jacek

    2010-05-01

    Fine roots play a significant role in plant and ecosystem respiration (RS); therefore, understanding factors controlling that process is important both to advancing understanding and potentially in modelling carbon (C) budgets. However, very little is known about the extent to which ectomycorrhizal (ECM) identity may influence RS or the underlying chemistry that may determine those rates. In order to test these relationships, we examined RS, measured as O(2) consumption, of first-order ECM root tips of Pinus sylvestris L. and Quercus robur L. saplings in relation to their ECM fungal symbionts and associated nitrogen (N), C and non-structural carbohydrate concentrations. Roots of P. sylvestris were colonized by Rhizopogon roseolus, Tuber sp. 1 and an unknown species of Pezizales. Fungal species colonizing Q. robur roots were Hebeloma sp., Tuber sp. 2 and one unidentified ECM fungus described as Tuber-like based on ECM morphology. ECM RS rates for different host species were significantly different and more than 97% of the variation in RS within a host species was explained by ECM root tip N concentrations. This may indicate that some of the variability in fine root RS-N relationships observed between and within different host species or their functional groups may be related to intraspecific host species differences in root tip N concentration among ECM fungal associates.

  8. NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

    PubMed Central

    Vega, Sonia; Neira, Jose L.; Marcuello, Carlos; Lostao, Anabel; Abian, Olga; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data. PMID:23803659

  9. [Research Progress in the Core Proteins of the Classical Swine Fever Virus].

    PubMed

    Hou, Yuzhen; Zhao, Dantong; Liu, Guoying; He, Fan; Liu, Bin; Fu, Shaoyin; Hao, Yongqing; Zhang, Wenguang

    2015-09-01

    The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.

  10. Whole-tree dynamics of non-structural carbohydrate and nitrogen pools across different seasons and in response to girdling in two temperate trees.

    PubMed

    Mei, Li; Xiong, Yanmei; Gu, Jiacun; Wang, Zhengquan; Guo, Dali

    2015-02-01

    Despite extensive research on the seasonal dynamics of non-structural carbohydrate (NSC) and nitrogen (N) concentrations, the size and relative contributions of NSC and N pools across different tree organs are not well understood. We have measured the changes in NSC and N concentrations in leaves, branches, stems and all root branch orders at monthly intervals in control and girdled trees of larch (Larix gmelinii) and ash (Fraxinus mandshurica). The biomass of each plant compartment was also determined to calculate the size of the NSC and N pools. In both species, 13-37% of the NSC and N pools were mobilized at the beginning of the growing season. Among the mobilized pools, stems and non-absorptive roots (branch orders 4-9) acted as the largest NSC sources in larch and ash, respectively, while branches served as the largest N source in both species. After stem girdling, 22 and 50% of the root NSC stores in larch and ash, respectively, were mobilized to maintain root activities during the growing season. Tree mortality was observed 1 year after girdling, at which time there was still an abundant NSC pool in the roots. We conclude that (1) different storage organs differ in their contribution to new tissue growth at the beginning of the growing season and that those storage organs holding higher fractions of the NSC or N pool are not necessarily those which mobilize more NSC or N; (2) tree growth may not be limited by carbon (C) availability; (3) C storage in non-absorptive roots plays an important role in maintaining tree survival after the termination of photosynthate flow from aboveground sources.

  11. Genetic Analysis of Avian Influenza Viruses: Cocirculation of Avian Influenza Viruses with Allele A and B Nonstructural Gene in Northern Pintail (Anas acuta) Ducks Wintering in Japan.

    PubMed

    Jahangir, Alam; Ruenphet, Sakchai; Sultana, Nadia; Shoham, Dany; Takehara, Kazuaki

    2012-01-01

    The pandemic influenza virus strains of 1918 (H1N1), 1957 (H2N2), 1968 (H3N2), and 2009 (H1N1) have genes related to avian influenza viruses (AIVs). The nonstructural (NS) gene of AIVs plays a significant role in host-viral interaction. However, little is known about the degree of diversity of this gene in Northern pintail (Anas acuta) ducks wintering in Japan. This study describes characteristics of pintail-originated H1N1, H1N2, H1N3, H5N2, H5N3, H5N9, and H7N7 viruses. Most of the viruses were revealed to be avian strains and not related to pandemic and seasonal flu strains. Nevertheless, the NP genes of 62.5% (5/8) viruses were found closely related to a A/swine/Korea/C12/08, indicating exchange of genetic material and ongoing mammalian-linked evolution of AIVs. Besides, all the viruses, except Aomori/422/07 H1N1, contain PSIQSR∗GLF motif usually found in avian, porcine, and human H1 strains. The Aomori/422/07 H1N1 has a PSVQSR∗GLF motif identical to a North American strain. This findings linked to an important intercontinental, Asian-American biogeographical interface. Phylogenetically all the viruses were clustered in Eurasian lineage. Cocirculation of allele A and B (NS gene) viruses was evident in the study implying the existence of a wide reservoir of influenza A viruses in pintail wintering in Japan.

  12. Higher Flexibility and Better Immediate Spontaneous Correction May Not Gain Better Results for Nonstructural Thoracic Curve in Lenke 5C AIS Patients

    PubMed Central

    Zhang, Yanbin; Lin, Guanfeng; Wang, Shengru; Zhang, Jianguo; Shen, Jianxiong; Wang, Yipeng; Guo, Jianwei; Yang, Xinyu; Zhao, Lijuan

    2016-01-01

    Study Design. Retrospective study. Objective. To study the behavior of the unfused thoracic curve in Lenke type 5C during the follow-up and to identify risk factors for its correction loss. Summary of Background Data. Few studies have focused on the spontaneous behaviors of the unfused thoracic curve after selective thoracolumbar or lumbar fusion during the follow-up and the risk factors for spontaneous correction loss. Methods. We retrospectively reviewed 45 patients (41 females and 4 males) with AIS who underwent selective TL/L fusion from 2006 to 2012 in a single institution. The follow-up averaged 36 months (range, 24–105 months). Patients were divided into two groups. Thoracic curves in group A improved or maintained their curve magnitude after spontaneous correction, with a negative or no correction loss during the follow-up. Thoracic curves in group B deteriorated after spontaneous correction with a positive correction loss. Univariate analysis and multivariate analysis were built to identify the risk factors for correction loss of the unfused thoracic curves. Results. The minor thoracic curve was 26° preoperatively. It was corrected to 13° immediately with a spontaneous correction of 48.5%. At final follow-up it was 14° with a correction loss of 1°. Thoracic curves did not deteriorate after spontaneous correction in 23 cases in group A, while 22 cases were identified with thoracic curve progressing in group B. In multivariate analysis, two risk factors were independently associated with thoracic correction loss: higher flexibility and better immediate spontaneous correction rate of thoracic curve. Conclusion. Posterior selective TL/L fusion with pedicle screw constructs is an effective treatment for Lenke 5C AIS patients. Nonstructural thoracic curves with higher flexibility or better immediate correction are more likely to progress during the follow-up and close attentions must be paid to these patients in case of decompensation. Level of Evidence: 4

  13. Glycaemic and insulinaemic responses to feeding hay with different non-structural carbohydrate content in control and polysaccharide storage myopathy-affected horses.

    PubMed

    Borgia, L; Valberg, S; McCue, M; Watts, K; Pagan, J

    2011-12-01

    The aim of this study was to determine whether the glycaemic/insulinaemic responses to hay with non-structural carbohydrate (NSC, soluble carbohydrate) of 17% (HC), 10% (MC) or 4% (LC) differs in control horses and whether these responses differ between control and horses with polysaccharide storage myopathy (PSSM). Five clinically normal control horses and seven PSSM horses, all unfit and of Quarter Horse breeding (age 9.4 ± 3.4 years, body condition score range: 4.5-6). A crossover design compared the HC and LC hay, with horses randomly assigned to hay type for 5 days, and all horses fed the MC hay during washout, after which the diets were switched. Horses were fed 1.5% BW (as fed) divided into 2 feeding per day, no grain. On morning of the fifth day of each block (seventh day for washout), horses were given 0.5% BW in hay, blood was drawn before and every 30 min for 5 h after feeding, and the rate of intake was measured. Whole blood glucose and plasma insulin were measured. The intake rate was significantly higher for HC. In control horses, the insulin area under the curve (6891.7 ± 3524.2 HC vs. 1185.4 ± 530.2 LC) was significantly higher than LC. Polysaccharide storage myopathy horses had significantly higher glycaemic and insulinaemic responses to HC vs. LC, however; the magnitude of insulin response was lower and glucose response higher in PSSM vs. control horses. Results suggest that insulin responses can differ significantly with the NSC content of hay. Feeding hay with 17% NSC produces elevations in insulin that could be detrimental for PSSM horses.

  14. Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

    PubMed Central

    Chen, Ruidong; Zhao, Xue; Wang, Yongxiang; Xie, Youhua; Liu, Jing

    2017-01-01

    The apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family proteins bind RNA and single-stranded DNA, and create C-to-U base modifications through cytidine deaminase activity. APOBEC3G restricts human immunodeficiency virus 1 (HIV-1) infection by creating hypermutations in proviral DNA, while HIV-1-encoded vif protein antagonizes such restriction by targeting APOBEC3G for degradation. APOBEC3G also inhibits hepatitis B virus (HBV): APOBEC3G co-expression inhibits HBV replication and evidences exist indicating APOBEC3G-mediated HBV hypermutations in patients. HBV encodes a small non-structural X protein (HBx) with a recognized activating effect on HBV life cycle. In this work, we report the discovery that HBx selectively and dose-dependently decreases the protein level of co-expressed APOBEC3G in transfected Huh-7 cells. The effect was shown to take place post-translationally, but does not rely on protein degradation via proteasome or lysosome. Further work demonstrated that intracellular APOBEC3G is normally exported via exosome secretion and inhibition of exosome biogenesis causes retention of intracellular APOBEC3G. Finally, HBx co-expression specifically enhanced externalization of APOBEC3G via exosomes, resulting in decrease of intracellular APOBEC3G protein level. These data suggest the possibility that in addition to other mechanisms, HBx-mediated activation of HBV might also involve antagonizing of intracellular restriction factor APOBEC3G through promotion of its export. PMID:28098260

  15. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

    PubMed Central

    Ylösmäki, Leena; Fagerlund, Riku; Kuisma, Inka; Julkunen, Ilkka; Saksela, Kalle

    2016-01-01

    The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. PMID:27092521

  16. Porcine Reproductive and Respiratory Syndrome Virus nsp1α Inhibits NF-κB Activation by Targeting the Linear Ubiquitin Chain Assembly Complex.

    PubMed

    Jing, Huiyuan; Fang, Liurong; Ding, Zhen; Wang, Dang; Hao, Wenqi; Gao, Li; Ke, Wenting; Chen, Huanchun; Xiao, Shaobo

    2017-02-01

    Linear ubiquitination, a newly discovered posttranslational modification, is catalyzed by the linear ubiquitin chain assembly complex (LUBAC), which is composed of three subunits: one catalytic subunit HOIP and two accessory molecules, HOIL-1L and SHARPIN. Accumulating evidence suggests that linear ubiquitination plays a crucial role in innate immune signaling and especially in the activation of the NF-κB pathway by conjugating linear polyubiquitin chains to NF-κB essential modulator (NEMO, also called IKKγ), the regulatory subunit of the IKK complex. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide, is an ideal model to study the host's disordered inflammatory responses after viral infection. Here, we found that LUBAC-induced NF-κB and proinflammatory cytokine expression can be inhibited in the early phase of PRRSV infection. Screening the PRRSV-encoded proteins showed that nonstructural protein 1α (nsp1α) suppresses LUBAC-mediated NF-κB activation and its CTE domain is required for the inhibition. Mechanistically, nsp1α binds to HOIP/HOIL-1L and impairs the interaction between HOIP and SHARPIN, thus reducing the LUBAC-dependent linear ubiquitination of NEMO. Moreover, PRRSV infection also blocks LUBAC complex formation and NEMO linear-ubiquitination, the important step for transducing NF-κB signaling. This unexpected finding demonstrates a previously unrecognized role of PRRSV nsp1α in modulating LUBAC signaling and explains an additional mechanism of immune modulation by PRRSV.

  17. Comparisons by peptide mapping of proteins specified by Kunjin, West Nile and Murray Valley encephalitis viruses.

    PubMed

    Wright, P J; Warr, H M; Westaway, E G

    1983-12-01

    The relationships among virus-specified proteins of Murray Valley encephalitis (MVE), Kunjin (KUN) and West Nile (WN) viruses were investigated by peptide mapping of exhaustive proteolytic digests of radioactively labelled polypeptides. Maps of the three structural proteins (E, C and M) derived from purified virions and of two non-structural proteins (NV5 and NV4) obtained from infected cells were compared. For each polypeptide considered, the peptide maps of the KUN and WN virus-specified proteins were more similar to each other than either was to the map of the corresponding MVE virus-specified protein. Since the polypeptides considered together account for approximately 60% of the coding capacity of the flavivirus genome, our results suggested that, for the three viruses examined, the genomes of KUN and WN viruses are the most closely related.

  18. Interannual and seasonal dynamics, and the age, of nonstructural carbohydrate pools in the stemwood of temperate trees across a climatic gradient in New England

    NASA Astrophysics Data System (ADS)

    Richardson, A. D.; Carbone, M. S.; Czimczik, C. I.; Keenan, T. F.; Schaberg, P.; Xu, X.

    2011-12-01

    Like all plants, forest trees accumulate and store surplus mobile carbon (C) compounds as resources to be used to support future growth. This can be viewed as a bet-hedging strategy, providing reserves that the tree can draw on in times of stress-e.g., following disturbance, disease, or extreme climatic events. In the context of climate change, understanding factors influencing the availability of these stored C compounds to support growth and metabolism is essential for predicting the resilience of forests to environmental stress factors. We conducted this study to investigate the role of these stored C pools in the context ecosystem C balance at time scales from days to years. At quarterly intervals over a three year period, we monitored stemwood total nonstructural carbohydrate (TNC) concentrations of the dominant tree species of New England. Work was conducted at three sites along a climatic gradient: an oak-dominated transition hardwood forest (Harvard Forest), a maple-beech-birch northern hardwood forest (Bartlett Experimental Forest), and a spruce-fir forest (Howland Forest). We observed large differences among species both in TNC concentrations, and in how the TNC pool is partitioned to different compounds (starch, sucrose, glucose, fructose, raffinose, xylose and stachyose). Within a species, however, seasonal dynamics were remarkably similar across sites. The interannual variability in maximum TNC concentrations appears to be smaller than interannual variability in annual net ecosystem exchange of CO2. With an additional set of samples, we are using the bomb radiocarbon (14C) spike to estimate the average age of the sugars and starches in the TNC pool, and relating this to factors such as size, age, and recent growth rates of each tree. Initial results suggest that these TNC pools range in age from several years to several decades old. The average ages of starch and sugar pools are related, with the starches generally being older than sugars

  19. Interannual and seasonal dynamics, and the age, of nonstructural carbohydrate pools in the stemwood of temperate trees across a climatic gradient in the Northeastern US

    NASA Astrophysics Data System (ADS)

    Richardson, A. D.; Carbone, M. S.; Czimczik, C. I.; Keenan, T. F.; Schaberg, P.; Murakami, P.; Xu, X.

    2012-04-01

    Like all plants, forest trees accumulate and store non-structural carbohydrates (NSC) as resources to be used in the future. This can be viewed as a bet-hedging strategy, providing reserves that the tree can draw on in times of stress—e.g., following disturbance, disease, or extreme climatic events. In the context of climate change, understanding factors influencing the availability of these stored NSC compounds to support growth and metabolism is essential for predicting the resilience of forests to environmental stress factors. We conducted this study to investigate the role of these stored NSC pools in the context of ecosystem C balance at time scales from days to years. At quarterly intervals over a three-year period, we monitored stemwood total NSC concentrations of the dominant tree species of New England. Work was conducted at three sites along a climatic gradient: an oak-dominated transition hardwood forest (Harvard Forest), a maple-beech-birch northern hardwood forest (Bartlett Experimental Forest), and a spruce-fir forest (Howland Forest). We observed large differences among species both in NSC concentrations, and in how the NSC pool is partitioned to different compounds (starch, sucrose, glucose, fructose, raffinose, and stachyose). Within a species, however, seasonal dynamics were remarkably similar across sites. We used the bomb radiocarbon (14C) spike to estimate the average age of the sugars and starches in the NSC pool in a subset of nine maple trees from each site. We found that the age of sugars ranged from 1-24 y and starches from 1-31 y. The ages of sugar and starch pools were highly correlated across all sites, and there was no significant difference in the mean age of the two pools, which was ~11 y. Using a one-pool representation of NSC reserves (similar to the standard approach used in several existing forest C models) our model FöBAAR (FOrest Biomass, Allocation, Assimilation and Respiration) failed to reproduce the seasonal NSC dynamics

  20. [Influence of mulching management on the relationships between foliar non-structural carbohydrates and N, P concentrations in Phyllostachys violascens stand].

    PubMed

    Guo, Zi-wu; Hu, Jun-jing; Yang, Qing-ping; Li, Ying-chun; Chen, Shuang-lin; Chen, Wei-jun

    2015-04-01

    To understand the physiological adaptive mechanism of Phyllostachys violascens to intensive mulching management, the effect of mulching management (CK, 1, 3 and 6 years) on the concentrations and ratios of non-structural carbohydrates (NSC), nitrogen (N) and phosphorus (P) in bamboo foliage, and their stoichiometry was investigated. The results showed the concentrations of NSC and soluble sugar increased, while the starch content and N/P decreased markedly in bamboo stand with 1-year mulching, compared to CK stand, which suggested the N limitation to bamboo growth was strengthened. Foliar soluble sugar content decreased significantly, while the starch content increased dramatically, and the NSC content by per unit mass of N and P reached the maximum in the bamboo stand with 3-year mulching, compared to all other treatments. Foliar NSC and soluble sugar contents decreased significantly, while foliar starch content and N/P increased dramatically in the stand with 6-year mulching, which suggested the P limitation to bamboo growth was strengthened. Foliar NSC content was positively correlated with N and P concentrations in a short-term mulching management stand (≤ 3 years), while showed negative relationship with N/P. The foliar starch content in the stand with 6-year mulching was negatively correlated with N and P contents, while was positively correlated with N/P. The results indicated that short-term mulching management accelerated the accumulation of soluble sugar and decomposition of starch in foliage, thus the growth and activity of Ph. violascens was enhanced greatly. Long-term mulching management promoted the starch accumulation, which led to the transition from N limitation to P limitation for bamboo growth. In summary, long-term (6 years) mulching management caused the decrease of growth and activity of Ph. violascens dramatically, thus enhancing the bamboo stand degradation. The utilization efficiency of N and P reached the highest in the stand with 3-year

  1. Internal management of non-structural carbohydrate resources in apple leaves and branch wood under a broad range of sink and source manipulations.

    PubMed

    Naschitz, Shaul; Naor, Amos; Genish, Shani; Wolf, Shmuel; Goldschmidt, Eliezer E

    2010-06-01

    Apple (Malus x domestica Borkh.) grown in a Mediterranean climate depends on regular irrigation throughout the growing season. The objective of the current study was to elucidate the changes in carbohydrate storage and utilization by mature, field-grown apple trees in response to water availability to the trees and to the level of cropping. Fourteen-year-old apple trees cv. 'Golden Delicious' were grown under various combinations of irrigation rate (11, 33 or 77 l day(-)(1) per tree) and crop level ( approximately 100, approximately 300 or >1000 fruits per tree) beginning 47 days after full bloom (DAFB). Non-structural carbohydrate concentrations were measured at 78 (leaves and branch wood), 102 (leaves), 183 (branch wood) and 214 (branch wood) DAFB. Midday stem water potential (SWP) was measured at 2-week intervals between June and October. Trunk cross-sectional area was measured 47 and 265 DAFB. At harvest, 139 DAFB, the fruits of each tree were counted and weighed. SWP at 102 DAFB ranged between -0.6 and -2.7 MPa. Fruit fresh weight at harvest was positively related to SWP measured 37 days before harvest with distinct slopes for light/intermediate and heavy crop levels. Leaf and branch wood starch concentrations 78 and 102 DAFB were positively related to irrigation rate and negatively related to crop level. Mean fruit weight at harvest was positively related to branch wood starch concentration and neared maximum at a concentration of 40 mg g(-)(1) dry weight. Branch wood starch concentration recovered after harvest, especially in water-stressed trees. Sorbitol concentration was negatively related to irrigation rate. The sorbitol-to-starch concentration ratio in leaves at 102 DAFB was closely proportional to SWP. It is suggested that branch wood starch concentration represents the overall balance between carbon sources and sinks and may therefore serve as a reliable indicator of photo-assimilate availability. In water-stressed trees, sorbitol is prioritized over

  2. Nonstructural carbon dynamics are best predicted by the combination of photosynthesis and plant hydraulics during both bark beetle induced mortality and herbaceous plant response to drought

    NASA Astrophysics Data System (ADS)

    Ewers, B. E.; Mackay, D. S.; Guadagno, C.; Peckham, S. D.; Pendall, E.; Borkhuu, B.; Aston, T.; Frank, J. M.; Massman, W. J.; Reed, D. E.; Yarkhunova, Y.; Weinig, C.

    2012-12-01

    Recent work has shown that nonstructural carbon (NSC) provides both a signal and consequence of water stress in plants. The dynamics of NSC are likely not solely a result of the balance of photosynthesis and respiration (carbon starvation hypothesis) but also the availability of NSC for plant functions due to hydraulic condition. Further, plant hydraulics regulates photosynthesis both directly through stomatal conductance and indirectly through leaf water status control over leaf biochemistry. To test these hypotheses concerning NSC in response to a wide variety of plant perturbations, we used a model that combines leaf biochemical controls over photosynthesis (Farquhar model) with dynamic plant hydraulic conductance (Sperry model). This model (Terrestrial Regional Ecosystem Exchange Simulator; TREES) simulates the dynamics of NSC through a carbon budget approach that responds to plant hydraulic status. We tested TREES on two dramatically different datasets. The first dataset is from lodgepole pine and Engelmann spruce trees dying from bark beetles that carry blue-stain fungi which block xylem and cause hydraulic failure. The second data set is from Brassica rapa, a small herbaceous plant whose accessions are used in a variety of crops. The Brassica rapa plants include two parents whose circadian clock periods are different; NSC is known to provide inputs to the circadian clock likely modified by drought. Thus, drought may interact with clock control to constrain how NSC changes over the day. The Brassica rapa plants were grown in growth chamber conditions where drought was precisely controlled. The connection between these datasets is that both provide rigorous tests of our understanding of plant NSC dynamics and use similar leaf and whole plant gas exchange and NSC laboratory methods. Our results show that NSC decline (<10% in the whole plant) is less precipitous than expected from carbon starvation alone because both C uptake and use are impacted by water stress

  3. Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain.

    PubMed

    Kumar, Priti; Krishna, Venkatramana D; Sulochana, Paramadevanapalli; Nirmala, Gejjehalli; Haridattatreya, Maganti; Satchidanandam, Vijaya

    2004-02-01

    Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4(+) and CD8(+) T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

  4. Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    PubMed Central

    Chinnakannan, Senthil K.; Nanda, Sambit K.; Baron, Michael D.

    2013-01-01

    Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV) and measles virus (MeV) have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFNα/β) and type II (IFNγ) interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV), measles virus (MeV), peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action. PMID:23431397

  5. The Development of Nonstructural Alternatives.

    DTIC Science & Technology

    1979-05-01

    brief, such efforts can achieve little in the way of reducing damages to permanent improvements or hard to move possessions. Thus, in flash flood areas...radios could be supplied, bv various means, to all dwellings and/or business establishments and keyed to respond to flash flood warnings as well. b...Pred i cting the behavior of people -irn,, of an impending flash flood is much more difficult than predict- ;7ag tnle reliab-ility iind timeliness of

  6. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

    PubMed Central

    Alves, Rúbens Prince dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta

    2016-01-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

  7. Comparison of proteins specified by Murray Valley encephalitis, West Nile, Japanese encephalitis and St. Louis encephalitis viruses.

    PubMed

    Wright, P J; Warr, H M

    1986-10-01

    The relationships among proteins specified by Murray Valley encephalitis (MVE), West Nile (WN), Japanese encephalitis (JE) and St. Louis encephalitis (SLE) viruses were examined by peptide mapping. [3H]methionine-labelled tryptic peptides of viral proteins were separated by reverse phase high performance liquid chromatography (HPLC) and the separation profiles for a given protein specified by the different viruses were compared. Analyses of the non-structural protein NV5 (P98 or NS5) suggested that WN and SLE were the most closely related pair of viruses, and that JE was the virus most distant from the other three. Analyses of the structural proteins C and E failed to show the close relationship between WN and SLE indicated by the NV5 results, but did suggest that NV5 was the most conserved and E the least conserved of the three proteins.

  8. A global analysis of the concentration and dynamics of non-structural carbohydrates in plants: does it matter under global change? (Invited)

    NASA Astrophysics Data System (ADS)

    Sala, A.; Martínez-Vilalta, J.; Asencio, M.; Lloret, F.; Palacio, S.; Galiano, L.; Hoch, G.; Piper, F.

    2013-12-01

    Forests store significant amounts of C globally and recent reports of forest mortality world-wide have generated strong concern. Evidence suggests that increasing drought associated with climate change is a primary cause of tree stress and subsequent mortality. This has generated an urgent need to predict how forests will cope with increasing stress. Storage of non-structural C compounds (NSC, compounds not permanently invested in structural biomass that can later be used to support diverse plant functions) is critical for survival during periods when C assimilation does not meet demand. However, remarkable knowledge gaps exist to accurately predict plant growth and survival under climate change. Although trees accumulate relatively large pools of NSC, there is a strong debate on how these pools build up over time. On the one hand, it is frequently assumed that the build- up of NSC in trees occurs when supply via photosynthesis exceeds overall demands. If so, the abundant NSC pools in trees reflect an overabundance of C in the long term. An alternative explanation is that trees regulate NSC storage to maintain sufficient pools to cope with asynchronies between demand and supply and with stresses that long lived plants inevitably experience during their life time. However, our understanding of whether and how trees regulate storage in the long term is minimal. Here, we assembled a new global database to examine broad patterns of seasonal NSC variation across organs, life forms and biomes, and the degree to which NSC storage is depleted in plants under a wide range of natural conditions. We compiled seasonal data (at least three measurements over a minimum of four months) for ca. 200 wild species under natural conditions. On average, NSC account for ca. 8-10% of dry plant biomass. NSC and starch concentrations do not vary significantly with biome, but soluble sugars (SS) in plants from Mediterranean biomes are higher than in temperate or tropical biomes. On average

  9. Detecting molecular features of spectra mainly associated with structural and non-structural carbohydrates in co-products from bioEthanol production using DRIFT with uni- and multivariate molecular spectral analyses.

    PubMed

    Yu, Peiqiang; Damiran, Daalkhaijav; Azarfar, Arash; Niu, Zhiyuan

    2011-01-01

    The objective of this study was to use DRIFT spectroscopy with uni- and multivariate molecular spectral analyses as a novel approach to detect molecular features of spectra mainly associated with carbohydrate in the co-products (wheat DDGS, corn DDGS, blend DDGS) from bioethanol processing in comparison with original feedstock (wheat (Triticum), corn (Zea mays)). The carbohydrates related molecular spectral bands included: A_Cell (structural carbohydrates, peaks area region and baseline: ca. 1485-1188 cm(-1)), A_1240 (structural carbohydrates, peak area centered at ca. 1240 cm(-1) with region and baseline: ca. 1292-1198 cm(-1)), A_CHO (total carbohydrates, peaks region and baseline: ca. 1187-950 cm(-1)), A_928 (non-structural carbohydrates, peak area centered at ca. 928 cm(-1) with region and baseline: ca. 952-910 cm(-1)), A_860 (non-structural carbohydrates, peak area centered at ca. 860 cm(-1) with region and baseline: ca. 880-827 cm(-1)), H_1415 (structural carbohydrate, peak height centered at ca. 1415 cm(-1) with baseline: ca. 1485-1188 cm(-1)), H_1370 (structural carbohydrate, peak height at ca. 1370 cm(-1) with a baseline: ca. 1485-1188 cm(-1)). The study shows that the grains had lower spectral intensity (KM Unit) of the cellulosic compounds of A_1240 (8.5 vs. 36.6, P < 0.05), higher (P < 0.05) intensities of the non-structural carbohydrate of A_928 (17.3 vs. 2.0) and A_860 (20.7 vs. 7.6) than their co-products from bioethanol processing. There were no differences (P > 0.05) in the peak area intensities of A_Cell (structural CHO) at 1292-1198 cm(-1) and A_CHO (total CHO) at 1187-950 cm(-1) with average molecular infrared intensity KM unit of 226.8 and 508.1, respectively. There were no differences (P > 0.05) in the peak height intensities of H_1415 and H_1370 (structural CHOs) with average intensities 1.35 and 1.15, respectively. The multivariate molecular spectral analyses were able to discriminate and classify between the corn and corn DDGS molecular

  10. Detecting Molecular Features of Spectra Mainly Associated with Structural and Non-Structural Carbohydrates in Co-Products from BioEthanol Production Using DRIFT with Uni- and Multivariate Molecular Spectral Analyses

    PubMed Central

    Yu, Peiqiang; Damiran, Daalkhaijav; Azarfar, Arash; Niu, Zhiyuan

    2011-01-01

    The objective of this study was to use DRIFT spectroscopy with uni- and multivariate molecular spectral analyses as a novel approach to detect molecular features of spectra mainly associated with carbohydrate in the co-products (wheat DDGS, corn DDGS, blend DDGS) from bioethanol processing in comparison with original feedstock (wheat (Triticum), corn (Zea mays)). The carbohydrates related molecular spectral bands included: A_Cell (structural carbohydrates, peaks area region and baseline: ca. 1485–1188 cm−1), A_1240 (structural carbohydrates, peak area centered at ca. 1240 cm−1 with region and baseline: ca. 1292–1198 cm−1), A_CHO (total carbohydrates, peaks region and baseline: ca. 1187–950 cm−1), A_928 (non-structural carbohydrates, peak area centered at ca. 928 cm−1 with region and baseline: ca. 952–910 cm−1), A_860 (non-structural carbohydrates, peak area centered at ca. 860 cm−1 with region and baseline: ca. 880–827 cm−1), H_1415 (structural carbohydrate, peak height centered at ca. 1415 cm−1 with baseline: ca. 1485–1188 cm−1), H_1370 (structural carbohydrate, peak height at ca. 1370 cm−1 with a baseline: ca. 1485–1188 cm−1). The study shows that the grains had lower spectral intensity (KM Unit) of the cellulosic compounds of A_1240 (8.5 vs. 36.6, P < 0.05), higher (P < 0.05) intensities of the non-structural carbohydrate of A_928 (17.3 vs. 2.0) and A_860 (20.7 vs. 7.6) than their co-products from bioethanol processing. There were no differences (P > 0.05) in the peak area intensities of A_Cell (structural CHO) at 1292–1198 cm−1 and A_CHO (total CHO) at 1187–950 cm−1 with average molecular infrared intensity KM unit of 226.8 and 508.1, respectively. There were no differences (P > 0.05) in the peak height intensities of H_1415 and H_1370 (structural CHOs) with average intensities 1.35 and 1.15, respectively. The multivariate molecular spectral analyses were able to discriminate and classify between the corn and corn

  11. Separation and isolation of BTV dsRNA segments and viral proteins.

    PubMed

    Li, Joseph K-K; Huang, I-Jen; Hayama, Emiko

    2012-05-01

    Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

  12. Structure and function of the Zika virus full-length NS5 protein.

    PubMed

    Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; Chuang, Yin-Chih; Vaughan, Robert C; Sankaran, Banumathi; Kao, C Cheng; Li, Pingwei

    2017-03-27

    The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.

  13. Structure and function of the Zika virus full-length NS5 protein

    PubMed Central

    Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; Chuang, Yin-Chih; Vaughan, Robert C.; Sankaran, Banumathi; Kao, C. Cheng; Li, Pingwei

    2017-01-01

    The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication. PMID:28345656

  14. Assessing ubiquitination of viral proteins: lessons from flavivirus NS5

    PubMed Central

    Taylor, R. Travis; Best, Sonja M.

    2011-01-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains. PMID:21855635

  15. Assessing ubiquitination of viral proteins: Lessons from flavivirus NS5.

    PubMed

    Taylor, R Travis; Best, Sonja M

    2011-10-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains.

  16. Mitochondrial antiviral signaling adaptor mediated apoptosis in H3N2 swine influenza virus infection is inhibited by viral protein NS1 in vitro.

    PubMed

    Zhang, Jinqiu; Miao, Jinfeng; Hou, Jibo; Lu, Chengping

    2015-05-15

    We investigated the in vitro role of mitochondrial antiviral signaling adaptor (MAVS) in apoptosis induced by H3N2 swine influenza virus infection and the influence of viral NS1 (nonstructural protein 1) protein on this process. H3N2 swine influenza virus (SIV, A/Swine/Shandong/3/2005) was co-cultured with human lung epithelial A549 cells. The relationship of MAVS expression to SIV replication and apoptosis, and the influence of viral proteins on MAVS functions were studied. The data indicate that in response to SIV infection, MAVS was significantly upregulated at both the transcriptional and protein levels in the early stages of infection. Its expression and localization to mitochondria are necessary for apoptosis of epithelial cells induced by H3N2 swine influenza virus. Viral protein NS1 can antagonize MAVS-mediated apoptosis. These findings indicate that MAVS have a role in regulating innate mitochondrial responses to viral infection.

  17. Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

    PubMed

    Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

    2015-01-01

    Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

  18. The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein.

    PubMed Central

    Donald, R G; Lawrence, D M; Jackson, A O

    1997-01-01

    The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered. PMID:8995680

  19. Characterization of influenza virus variants with different sizes of the non-structural (NS) genes and their potential as live influenza vaccine in poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The influenza virus isolate A/turkey/Oregon/71-delNS1 (H7N3) has a 10 nucleotide deletion in the coding region of the NS1 gene and as a result produces a truncated NS1 protein. From a stock of this virus, we found that several variants with different sizes of the NS genes exist. The number of varian...

  20. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection

    PubMed Central

    Bird, Brian H.; Albariño, Cesar G.; Nichol, Stuart T.

    2007-01-01

    Rift Valley fever (RVF) virus belongs to the Bunyaviridae family of segmented negative-strand RNA viruses and causes mosquito-borne disease in sub-Saharan Africa. We report the development of a T7 RNA polymerase driven plasmid-based genetic system for the virulent Egyptian isolate, ZH501. We have used this system to rescue a virus that has a 387 nucleotide deletion on the genomic M segment that eliminates the coding region for two non-structural proteins known as NSm. This virus, ΔNSm rZH501, is indistinguishable from the parental ZH501 strain with respect to expression of structural proteins and growth in cultured mammalian cells. PMID:17070883

  1. The Molecular Mechanisms Underlying the Hijack of Host Proteins by the 1918 Spanish Influenza Virus.

    PubMed

    Shen, Qingliang; Zeng, Danyun; Zhao, Baoyu; Bhatt, Veer S; Li, Pingwei; Cho, Jae-Hyun

    2017-04-05

    The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.

  2. Splicing of the large intron present in the nonstructural gene of minute virus of mice is governed by TIA-1/TIAR binding downstream of the nonconsensus donor.

    PubMed

    Choi, Eun-Young; Pintel, David

    2009-06-01

    The essential proteins NS1 and NS2 of minute virus of mice are encoded by mRNAs R1 and R2, respectively. R2 is derived from R1 by excision of a large intron and thus splicing governs the relative ratios of NS1 and NS2. Excision of the large intron utilizes a nonconsensus 5' donor site. We identified a U-rich and A-rich intronic sequence immediately downstream of the nonconsensus 5' donor site that functions as an intronic splicing enhancer (ISE) required for efficient large-intron excision. The ISE binds the cellular RNA-processing proteins TIA-1 and TIAR, which enhance usage of the nonconsensus donor.

  3. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    PubMed

    Hedil, Marcio; Kormelink, Richard

    2016-07-23

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  4. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

    PubMed Central

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

  5. ABA and GA3 increase carbon allocation in different organs of grapevine plants by inducing accumulation of non-structural carbohydrates in leaves, enhancement of phloem area and expression of sugar transporters.

    PubMed

    Murcia, Germán; Pontin, Mariela; Reinoso, Herminda; Baraldi, Rita; Bertazza, Gianpaolo; Gómez-Talquenca, Sebastián; Bottini, Rubén; Piccoli, Patricia N

    2016-03-01

    Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A3 (GA3) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot-grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA3 solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre-veraison, full veraison and post-veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA-treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA3 increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA3 to berries and stems, respectively, was due to build-up of non-structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.

  6. Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5.

    PubMed

    Upadhyay, Anup K; Cyr, Matthew; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J

    2017-03-01

    The rapid spread of the recent Zika virus (ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 of Zika virus (ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space group P21212 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein from Japanese encephalitis virus and suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.

  7. Prediction of protein-protein interaction sites using an ensemble method

    PubMed Central

    2009-01-01

    Background Prediction of protein-protein interaction sites is one of the most challenging and intriguing problems in the field of computational biology. Although much progress has been achieved by using various machine learning methods and a variety of available features, the problem is still far from being solved. Results In this paper, an ensemble method is proposed, which combines bootstrap resampling technique, SVM-based fusion classifiers and weighted voting strategy, to overcome the imbalanced problem and effectively utilize a wide variety of features. We evaluate the ensemble classifier using a dataset extracted from 99 polypeptide chains with 10-fold cross validation, and get a AUC score of 0.86, with a sensitivity of 0.76 and a specificity of 0.78, which are better than that of the existing methods. To improve the usefulness of the proposed method, two special ensemble classifiers are designed to handle the cases of missing homologues and structural information respectively, and the performance is still encouraging. The robustness of the ensemble method is also evaluated by effectively classifying interaction sites from surface residues as well as from all residues in proteins. Moreover, we demonstrate the applicability of the proposed method to identify interaction sites from the non-structural proteins (NS) of the influenza A virus, which may be utilized as potential drug target sites. Conclusion Our experimental results show that the ensemble classifiers are quite effective in predicting protein interaction sites. The Sub-EnClassifiers with resampling technique can alleviate the imbalanced problem and the combination of Sub-EnClassifiers with a wide variety of feature groups can significantly improve prediction performance. PMID:20015386

  8. The herpes simplex virus UL37 protein is phosphorylated in infected cells.

    PubMed Central

    Albright, A G; Jenkins, F J

    1993-01-01

    The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation. Images PMID:8392618

  9. Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity▿

    PubMed Central

    Jakubiec, Anna; Drugeon, Gabrièle; Camborde, Laurent; Jupin, Isabelle

    2007-01-01

    Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA. PMID:17686855

  10. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    SciTech Connect

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  11. Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

    PubMed Central

    Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

    1998-01-01

    NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

  12. Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

    PubMed Central

    Haatveit, Hanne Merethe; Wessel, Øystein; Markussen, Turhan; Lund, Morten; Thiede, Bernd; Nyman, Ingvild Berg; Braaen, Stine; Dahle, Maria Krudtaa; Rimstad, Espen

    2017-01-01

    Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection. PMID:28335455

  13. A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

    PubMed Central

    Dodes Traian, Martín M.; Cattoni, Diego I.; Levi, Valeria; González Flecha, F. Luis

    2012-01-01

    Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes. PMID:22723977

  14. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  15. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  16. Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated tra...

  17. Expression of Aleutian mink disease parvovirus proteins in a baculovirus vector system.

    PubMed Central

    Christensen, J; Storgaard, T; Bloch, B; Alexandersen, S; Aasted, B

    1993-01-01

    We have previously published a detailed transcription map of Aleutian mink disease parvovirus (ADV) and proposed a model for the translation of the two virion structural proteins (VP1 and VP2) and three nonstructural proteins (NS-1, NS-2, and NS-3) (S. Alexandersen, M. E. Bloom, and S. Perryman, J. Virol. 62:3684-3994, 1988). To verify and further characterize this model, we cloned the predicted open reading frames for NS-1, NS-2, NS-3, VP1-VP2, and VP2 alone into a recombinant baculovirus and expressed them in Sf9 insect cells. Expression of VP1-VP2 or VP2 alone in cDNA and in the genomic form was achieved. The expressed proteins had molecular weights similar to those of the corresponding proteins of wild-type ADV-G, although the ratio of VP1 to VP2 was altered. The recombinant baculovirus-expressed ADV VP1 and VP2 showed nuclear localization in Sf9 cells and were able to form particles indistinguishable, by electron microscopy, from wild-type virus. The large nonstructural protein, NS-1, showed predominantly nuclear localization in Sf9 cells when analyzed by immunofluorescence and had a molecular weight similar to that of wild-type ADV NS-1. Moreover, expression of NS-1 in Sf9 cells caused a change in morphology of the cells and resulted in 10-times-lower titers of recombinant baculovirus during infection, suggesting a cytostatic or cytotoxic action of this protein. The smaller NS-2 gene product seems to be located in the cytoplasm. When analyzed by Western immunoblotting, NS-2 comigrated with an approximately 16-kDa band seen in lysates of ADV-infected feline kidney cells. The putative NS-3 gene product exhibited a diffuse distribution in Sf9 cells and had a molecular weight of approximately 10,000. All of the expressed ADV-encoded proteins were recognized by sera from ADV-infected mink. Thus, expression of ADV cDNAs allowed assignment of the different mRNAs to the viral proteins observed during ADV infection in cell culture and supported our previously proposed

  18. Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein.

    PubMed

    Yakshe, Krystle A; Franklin, Zachary D; Ball, Judith M

    2015-05-01

    Rotavirus (RV) contains 11 double-stranded RNA segments that encode for twelve structural and nonstructural proteins. The separation and isolation of viral RNA is a necessary precursor for many experimental techniques and can be useful for rapid RV RNA typing and sequencing of different rotavirus strains. The segmented genome enables RV to recombine easily. These recombinant viruses are essential for many purposes, including generation of potential vaccine strains. Rotavirus gene 10 expresses the viral enterotoxin, NSP4, which has been the focus of several studies due to the influence of NSP4 on rotavirus replication, morphogenesis, and pathogenesis. This unit will describe the isolation and separation of viral RNAs, the production characterization of recombinant RV in culture, and the expression and isolation of NSP4 in mammalian and insect cells.

  19. Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions▿

    PubMed Central

    Martró, Elisa; González, Victoria; Buckton, Andrew J.; Saludes, Verónica; Fernández, Gema; Matas, Lurdes; Planas, Ramón; Ausina, Vicenç

    2008-01-01

    We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5′ noncoding region (5′NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5′NC reverse hybridization (method 2; InnoLiPA HCV II) and 5′NC sequencing (method 3; Trugene HCV 5′NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays. PMID:17989191

  20. Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein

    USGS Publications Warehouse

    Fassbinder-Orth, C. A.; Hofmeister, E.K.; Weeks-Levy, C.; Karasov, W.H.

    2009-01-01

    West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 ??g or 100 ??g/dose), of E protein intramuscularly (IM, 20 ??g/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds. ?? 2009 American Association of Avian Pathologists.

  1. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure.

    PubMed

    Urbaczek, Ana Carolina; Ribeiro, Lívia Carolina de Abreu; Ximenes, Valdecir Farias; Afonso, Ana; Nogueira, Camila Tita; Generoso, Wesley Cardoso; Alberice, Juliana Vieira; Rudnicki, Martina; Ferrer, Renila; Fonseca, Luiz Marcos da; Costa, Paulo Inácio da

    2014-09-01

    The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

  2. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins

    PubMed Central

    Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; Sankaran, Banumathi; Du, Fenglei; Shelton, Catherine L.; Herr, Andrew B.; Ji, Jun-Yuan; Li, Pingwei

    2016-01-01

    Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. PMID:27302953

  3. Mapping the interactions of dengue virus NS1 protein with human liver proteins using a yeast two-hybrid system: identification of C1q as an interacting partner.

    PubMed

    Silva, Emiliana M; Conde, Jonas N; Allonso, Diego; Nogueira, Mauricio L; Mohana-Borges, Ronaldo

    2013-01-01

    Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection.

  4. Transient protein-protein interface prediction: datasets, features, algorithms, and the RAD-T predictor

    PubMed Central

    2014-01-01

    Background Transient protein-protein interactions (PPIs), which underly most biological processes, are a prime target for therapeutic development. Immense progress has been made towards computational prediction of PPIs using methods such as protein docking and sequence analysis. However, docking generally requires high resolution structures of both of the binding partners and sequence analysis requires that a significant number of recurrent patterns exist for the identification of a potential binding site. Researchers have turned to machine learning to overcome some of the other methods’ restrictions by generalising interface sites with sets of descriptive features. Best practices for dataset generation, features, and learning algorithms have not yet been identified or agreed upon, and an analysis of the overall efficacy of machine learning based PPI predictors is due, in order to highlight potential areas for improvement. Results The presence of unknown interaction sites as a result of limited knowledge about protein interactions in the testing set dramatically reduces prediction accuracy. Greater accuracy in labelling the data by enforcing higher interface site rates per domain resulted in an average 44% improvement across multiple machine learning algorithms. A set of 10 biologically unrelated proteins that were consistently predicted on with high accuracy emerged through our analysis. We identify seven features with the most predictive power over multiple datasets and machine learning algorithms. Through our analysis, we created a new predictor, RAD-T, that outperforms existing non-structurally specializing machine learning protein interface predictors, with an average 59% increase in MCC score on a dataset with a high number of interactions. Conclusion Current methods of evaluating machine-learning based PPI predictors tend to undervalue their performance, which may be artificially decreased by the presence of un-identified interaction sites. Changes to

  5. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    SciTech Connect

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  6. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

    PubMed Central

    Stinski, M F

    1978-01-01

    At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication. Images PMID:209215

  7. Human heart cell proteins interacting with a C-terminally truncated 2A protein of coxsackie B3 virus: identification by the yeast two-hybrid system.

    PubMed

    Zhao, Tiansheng; Huang, Xiaotian; Xia, Yanhua

    2016-04-01

    Protein 2A is a non-structural protein of coxsackievirus B3 (CVB3), an important human pathogen that can cause a variety of human diseases. Protein 2A not only participates in viral life cycle, but also regulates host cell functions; however, the underlying mechanisms remain poorly understood. In order to better understand the molecular mechanisms of CVB3 2A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CVB3 2A interactive proteins in the human heart cDNA library. Full-length 2A shows strong transcriptional activity in yeast cells, which interferes with the application of Y2H system; therefore, a series of 2A deletion mutants were constructed. Analysis of transcriptional self-activation revealed that 2A lost its transcriptional activity after truncation of 60 amino acids (aa) at the N-terminus or deletion of 17 aa at the C-terminus. Choosing the 2A mutant with 17 aa deletion at the C-terminus as the bait protein, four interactive cellular proteins were identified, including TIMP4, MYL2, COX7C, and ENO1. These proteins are mostly related to protein degradation and metabolism. Although the interactions detected by the Y2H system should be considered as preliminary results, the finding of proteins translated from a human heart cDNA library that interacts with the CVB3 2A will stimulate experiments testing the reactivity of a translational mixture derived from that library with full-length 2A protein, followed by co-immunoprecipitation studies.

  8. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  9. Identification of feline panleukopenia virus proteins expressed in Purkinje cell nuclei of cats with cerebellar hypoplasia.

    PubMed

    Poncelet, Luc; Héraud, Céline; Springinsfeld, Marie; Ando, Kunie; Kabova, Anna; Beineke, Andreas; Peeters, Dominique; Op De Beeck, Anne; Brion, Jean-Pierre

    2013-06-01

    Parvoviruses depend on initiation of host cell division for their replication. Undefined parvoviral proteins have been detected in Purkinje cells of the cerebellum after experimental feline panleukopenia virus (FPV) infection of neonatal kittens and in naturally occurring cases of feline cerebellar hypoplasia. In this study, a parvoviral protein in the nucleus of Purkinje cells of kittens with cerebellar hypoplasia was shown by immunoprecipitation to be the FPV viral capsid protein VP2. In PCR-confirmed, FPV-associated feline cerebellar hypoplasia, expression of the FPV VP2 protein was demonstrated by immunohistochemistry in Purkinje cell nuclei in 4/10 cases and expression of the FPV non-structural protein NS1 was demonstrated in Purkinje cell nuclei in 5/10 cases. Increased nuclear ERK1 expression was observed in several Purkinje cells in 1/10 kittens. No expression of the G1 and S mitotic phase marker proliferating cell nuclear antigen (PCNA) was evident in Purkinje cell nuclei. These results support the hypothesis that FPV is able to proceed far into its replication cycle in post-mitotic Purkinje cells.

  10. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    PubMed

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  11. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  12. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  13. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  14. Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry

    PubMed Central

    Chevin, Aurore; Coutard, Bruno; Blanchard, Philippe; Dabert-Gay, Anne-Sophie; Ribière-Chabert, Magali; Thiéry, Richard

    2015-01-01

    Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed. PMID:26110588

  15. Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry.

    PubMed

    Chevin, Aurore; Coutard, Bruno; Blanchard, Philippe; Dabert-Gay, Anne-Sophie; Ribière-Chabert, Magali; Thiéry, Richard

    2015-06-23

    Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.

  16. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    SciTech Connect

    Paterson, Carolyn P.; Ayalew, Lisanework E.; Tikoo, Suresh K.

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  17. The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.

    PubMed

    Tyagi, Shweta; Korkaya, Hasan; Zafrullah, Mohammad; Jameel, Shahid; Lal, Sunil K

    2002-06-21

    Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

  18. Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

    PubMed Central

    Ishida, C; Matsumoto, K; Fukada, K; Matsushita, K; Shiraki, H; Maeda, Y

    1993-01-01

    We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein. PMID:7681849

  19. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  20. Development of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against the 3B Protein of Foot-and-Mouth Disease Virus

    PubMed Central

    Yang, Ming; Parida, Satya; Salo, Tim; Hole, Kate; Velazquez-Salinas, Lauro

    2015-01-01

    Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins. PMID:25651918

  1. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2006-12-20

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

  2. Whey Protein

    MedlinePlus

    ... inflammation (polymyalgia rheumatica). Taking whey protein in a dairy product twice daily for 8 weeks does not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. More evidence is needed to rate whey protein for these uses.

  3. Serum concentration and increased temperature alter Mayaro virus RNA and protein synthesis in Aedes albopictus (mosquito)-infected cells.

    PubMed

    Motta, M C; Fournier, M V; Carvalho, M G

    1995-01-01

    We have previously shown the inhibition of Mayaro virus multiplication in Aedes albopictus-infected cells maintained at a supraoptimal temperature for growth (37 degrees C) and a stimulation of virus production in response to high serum concentrations in the incubation medium. In the present study, we addressed the question of how the effect of continuous heat stress and high serum concentration soon after infection interfere with virus macromolecule synthesis. Cells maintained at 28 degrees C in the presence of 2% serum synthesized a viral genomic RNA of 12 kb and a subgenomic RNA of 5.2 kb 6 h postinfection. Analysis of the protein profile showed the presence of the viral nucleocapsid protein of 34 kDa (P34). However, if infected cells were maintained at 37 degrees C, a smear starting immediately below the 5.2-kb RNA was noticed and the viral P34 was not detected by SDS-PAGE. Addition of 10% serum to the growth medium of infected cells maintained at 37 degrees C results in a viral RNA profile and protein synthesis similar to those observed in cultures kept at 28 degrees C, i.e., the smear was not observed and the P34 protein was detected. The results suggest that the inhibition of virus multiplication by temperature may be related to the inhibition of viral nonstructural protein synthesis early during infection. The presence of high serum levels in the incubation medium protects macromolecule synthesis against heat stress.

  4. Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

    SciTech Connect

    Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

    2008-07-18

    Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

  5. Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes

    PubMed Central

    Liu, Jianying; Liu, Yang; Nie, Kaixiao; Du, Senyan; Qiu, Jingjun; Pang, Xiaojing; Wang, Penghua; Cheng, Gong

    2016-01-01

    Summary The arbovirus life cycle involves viral transfer between a vertebrate host and an arthropod vector, and acquisition of virus from an infected mammalian host by a vector is an essential step in this process. Here, we report that flavivirus nonstructural protein-1 (NS1), which is abundantly secreted into the serum of an infected host, plays a critical role in flavivirus acquisition by mosquitoes. The presence of dengue virus (DENV) and Japanese encephalitis virus (JEV) NS1s in the blood of infected interferon alpha and gamma receptor-deficient mice (AG6) facilitated virus acquisition by their native mosquito vectors because the protein enabled the virus to overcome the immune barrier of the mosquito midgut. Active immunization of AG6 mice with a modified DENV NS1 reduced DENV acquisition by mosquitoes and protected mice against a lethal DENV challenge, suggesting that immunization with NS1 could reduce the number of virus-carrying mosquitoes as well as the incidence of flaviviral diseases. Our study demonstrates that flaviviruses utilize NS1 proteins produced during their vertebrate phases to enhance their acquisition by vectors, which might be a result of flavivirus evolution to adapt to multiple host environments. PMID:27562253

  6. Crystal Structure of the Avian Reovirus Inner Capsid Protein σA▿

    PubMed Central

    Guardado-Calvo, Pablo; Vazquez-Iglesias, Lorena; Martinez-Costas, José; Llamas-Saiz, Antonio L.; Schoehn, Guy; Fox, Gavin C.; Hermo-Parrado, X. Lois; Benavente, Javier; van Raaij, Mark J.

    2008-01-01

    Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural σA protein is a major component of the inner capsid, clamping together λA building blocks. σA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed σA by molecular replacement and refined it using data to 2.3-Å resolution. Twelve σA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of σA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of σA to double-stranded RNA. The minimal length of double-stranded RNA required for σA binding was observed to be 14 to 18 bp. PMID:18799570

  7. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

    PubMed

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana

    2008-06-01

    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  8. The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

    PubMed Central

    Riedel, Christiane; Lamp, Benjamin; Heimann, Manuela; König, Matthias; Blome, Sandra; Moennig, Volker; Schüttler, Christian; Thiel, Heinz-Jürgen; Rümenapf, Tillmann

    2012-01-01

    Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general. PMID:22457622

  9. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  10. Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.

    PubMed

    Chu, Derrick; Ren, Songyang; Hu, Stacy; Wang, Wei Gang; Subramanian, Aparna; Contreras, Deisy; Kanagavel, Vidhya; Chung, Eric; Ko, Justine; Amirtham Jacob Appadorai, Ranjit Singh; Sinha, Sanjeev; Jalali, Ziba; Hardy, David W; French, Samuel W; Arumugaswami, Vaithilingaraja

    2013-05-01

    Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.

  11. Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A.

    PubMed

    Kim, Nari; Kim, Min-Jung; Sung, Pil Soo; Bae, Yong Chul; Shin, Eui-Cheol; Yoo, Joo-Yeon

    2016-02-12

    Hepatitis C virus (HCV) utilizes autophagy to promote its propagation. Here we show the autophagy-mediated suppression of HCV replication via the endoplasmic reticulum (ER) protein SCOTIN. SCOTIN overexpression inhibits HCV replication and infectious virion production in cells infected with cell culture-derived HCV. HCV nonstructural 5A (NS5A) protein, which is a critical factor for HCV RNA replication, interacts with the IFN-β-inducible protein SCOTIN, which transports NS5A to autophagosomes for degradation. Furthermore, the suppressive effect of SCOTIN on HCV replication is impaired in both ATG7-silenced cells and cells treated with autophagy or lysosomal inhibitors. SCOTIN does not affect the overall flow of autophagy; however, it is a substrate for autophagic degradation. The physical association between the transmembrane/proline-rich domain (TMPRD) of SCOTIN and Domain-II of NS5A is essential for autophagosomal trafficking and NS5A degradation. Altogether, our findings suggest that IFN-β-induced SCOTIN recruits the HCV NS5A protein to autophagosomes for degradation, thereby restricting HCV replication.

  12. Analysis of proteins encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm - structural protein with DNA polymerase motif.

    PubMed

    Hayakawa, T; Kojima, K; Nonaka, K; Nakagaki, M; Sahara, K; Asano, S i; Iizuka, T; Bando, H

    2000-01-01

    Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.

  13. Protein assay structured on paper by using lithography

    NASA Astrophysics Data System (ADS)

    Wilhelm, E.; Nargang, T. M.; Al Bitar, W.; Waterkotte, B.; Rapp, B. E.

    2015-03-01

    There are two main challenges in producing a robust, paper-based analytical device. The first one is to create a hydrophobic barrier which unlike the commonly used wax barriers does not break if the paper is bent. The second one is the creation of the (bio-)specific sensing layer. For this proteins have to be immobilized without diminishing their activity. We solve both problems using light-based fabrication methods that enable fast, efficient manufacturing of paper-based analytical devices. The first technique relies on silanization by which we create a flexible hydrophobic barrier made of dimethoxydimethylsilane. The second technique demonstrated within this paper uses photobleaching to immobilize proteins by means of maskless projection lithography. Both techniques have been tested on a classical lithography setup using printed toner masks and on a lithography system for maskless lithography. Using these setups we could demonstrate that the proposed manufacturing techniques can be carried out at low costs. The resolution of the paper-based analytical devices obtained with static masks was lower due to the lower mask resolution. Better results were obtained using advanced lithography equipment. By doing so we demonstrated, that our technique enables fabrication of effective hydrophobic boundary layers with a thickness of only 342 μm. Furthermore we showed that flourescine-5-biotin can be immobilized on the non-structured paper and be employed for the detection of streptavidinalkaline phosphatase. By carrying out this assay on a paper-based analytical device which had been structured using the silanization technique we proofed biological compatibility of the suggested patterning technique.

  14. Cellular localization and effects of ectopically expressed hepatitis A virus proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC.

    PubMed

    Seggewiß, Nicole; Kruse, Hedi Verena; Weilandt, Rebecca; Domsgen, Erna; Dotzauer, Andreas; Paulmann, Dajana

    2016-04-01

    In the course of hepatitis A virus (HAV) infections, the seven nonstructural proteins and their intermediates are barely detectable. Therefore, little is known about their functions and mechanisms of action. Ectopic expression of the presumably membrane-associated proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC allowed the intracellular localization of these proteins and their possible function during the replication cycle of HAV to be investigated. In this study, we used rhesus monkey kidney cells, which are commonly used for cell culture experiments, and human liver cells, which are the natural target cells. We detected specific associations of these proteins with distinct membrane compartments and the cytoskeleton, different morphological alterations of the respective structures, and specific effects on cellular functions. Besides comparable findings in both cell lines used with regard to localization and effects of the proteins examined, we also found distinct differences. The data obtained identify so far undocumented interactions with and effects of the HAV proteins investigated on cellular components, which may reflect unknown aspects of the interaction of HAV with the host cell, for example the modification of the ERGIC (ER-Golgi intermediate compartment) structure, an interaction with lipid droplets and lysosomes, and inhibition of the classical secretory pathway.

  15. Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.

    PubMed

    Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jesús-Ortega, Nereyda; Santos-López, Gerardo; Vallejo-Ruiz, Verónica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio

    2014-06-24

    Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

  16. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  17. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  18. Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

    PubMed

    Hodgson, Teri; Britton, Paul; Cavanagh, Dave

    2006-01-01

    Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo.

  19. Bioengineered Vaults: Self-Assembling Protein Shell–Lipophilic Core Nanoparticles for Drug Delivery

    PubMed Central

    2015-01-01

    We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems. PMID:25061969

  20. ICP35 Is a TREX-Like Protein Identified in White Spot Syndrome Virus

    PubMed Central

    Phairoh, Panapat; Suthibatpong, Thana; Rattanarojpong, Triwit; Jongruja, Nujarin; Senapin, Saengchan; Choowongkomon, Kiattawee; Khunrae, Pongsak

    2016-01-01

    ICP35 is a non-structural protein from White spot syndrome virus believed to be important in viral replication. Since ICP35 was found to localize in the host nucleus, it has been speculated that the function of ICP35 might be involved in the interaction of DNA. In this study, we overexpressed, purified and characterized ICP35. The thioredoxin-fused ICP35 (thio-ICP35) was strongly expressed in E. coli and be able to form itself into dimers. Investigation of the interaction between ICP35 and DNA revealed that ICP35 can perform DNase activity. Structural model of ICP35 was successfully built on TREX1, suggesting that ICP35 might adopt the folding similar to that of TREX1 protein. Several residues important for dimerization in TREX1 are also conserved in ICP35. Residue Asn126 and Asp132, which are seen to be in close proximity to metal ions in the ICP35 model, were shown through site-directed mutagenesis to be critical for DNase activity. PMID:27348862

  1. Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

    SciTech Connect

    Cheng, A.; Wong, S; Yuan, Y

    2009-01-01

    Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

  2. Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

    PubMed

    de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

    2017-01-18

    We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KD(eff). Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

  3. A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

    PubMed Central

    Rostad, Christina A.; Stobart, Christopher C.; Gilbert, Brian E.; Pickles, Ray J.; Hotard, Anne L.; Meng, Jia; Blanco, Jorge C. G.; Moin, Syed M.; Graham, Barney S.; Piedra, Pedro A.

    2016-01-01

    ABSTRACT Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCE RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV

  4. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

    PubMed

    Costa, Simone M; Yorio, Anna Paula; Gonçalves, Antônio J S; Vidale, Mariana M; Costa, Emmerson C B; Mohana-Borges, Ronaldo; Motta, Marcia A; Freire, Marcos S; Alves, Ada M B

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

  5. A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

    PubMed Central

    Zhao, Yongqian; Soh, Tingjin Sherryl; Zheng, Jie; Chan, Kitti Wing Ki; Phoo, Wint Wint; Lee, Chin Chin; Tay, Moon Y. F.; Swaminathan, Kunchithapadam; Cornvik, Tobias C.; Lim, Siew Pheng; Shi, Pei-Yong; Lescar, Julien; Vasudevan, Subhash G.; Luo, Dahai

    2015-01-01

    Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5. PMID:25775415

  6. RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

    PubMed Central

    Vitour, Damien; Lindenbaum, Pierre; Vende, Patrice; Becker, Michelle M.; Poncet, Didier

    2004-01-01

    Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation. PMID:15047801

  7. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  8. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus.

    PubMed

    Avellaneda, Gloria; Mundt, Egbert; Lee, Chang-Won; Jadhao, Samadhan; Suarez, David L

    2010-03-01

    Vaccination against avian influenza (AI) virus, a powerful tool for control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater worldwide acceptance if a reliable test were available that clearly discriminated between naturally infected and vaccinated-only animals (DIVA). Because the nonstructural protein (NS1) is expressed in influenza virus-infected cells, and it is not packaged in the virion, it is an attractive candidate for a DIVA differential diagnostic test. The aim of this work was to determine the onset of the antibody response to the NS1 protein in chickens infected with low pathogenic avian influenza (LPAI) virus, and to evaluate the diagnostic potential of a baculovirus-expressed purified NS1 protein in an indirect ELISA-based DIVA strategy. An antibody response against NS1 was first detected 3 wk after infection, but the antibody levels were decreasing rapidly by 5 wk after infection. However, most chickens did not have detectable antibodies in spite of high hemagglutination inhibition (HI) antibody titers in one group. In birds vaccinated with inactivated oil-emulsion vaccines, antibodies against NS1 were not detected before virulent challenge, and only a small percentage of birds seroconverted after homologous LPAI virus challenge. Vaccinated birds challenged with highly pathogenic AI showed a higher NS1 antibody response, but at most only 40% of birds seroconverted against NS1 protein by 3 wk after challenge. Because of the variability of seroconversion and the duration of the antibody response in chickens, the NS1 protein DIVA strategy did not perform as well as expected, and if this strategy were to be used, it would require sampling a higher number of birds to compensate for the lower seroconversion rate.

  9. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

    PubMed

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L

    2015-08-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

  10. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

    PubMed Central

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

    2015-01-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

  11. The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter.

    PubMed Central

    Pereira, D J; Muzyczka, N

    1997-01-01

    Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA. PMID:9032303

  12. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    SciTech Connect

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  13. Development of an insect vector cell culture and RNA interference system to investigate the functional role of fijivirus replication protein.

    PubMed

    Jia, Dongsheng; Chen, Hongyan; Zheng, Ailing; Chen, Qian; Liu, Qifei; Xie, Lianhui; Wu, Zujian; Wei, Taiyun

    2012-05-01

    An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins.

  14. Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

    PubMed

    Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva; Mohana-Borges, Ronaldo

    2016-11-01

    Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system.

  15. Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector.

    PubMed

    Kuznetsova, Irina; Shurygina, Anna-Polina; Wolf, Brigitte; Wolschek, Markus; Enzmann, Florian; Sansyzbay, Abylay; Khairullin, Berik; Sandybayev, Nurlan; Stukova, Marina; Kiselev, Oleg; Egorov, Andrej; Bergmann, Michael

    2014-02-01

    The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

  16. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease.

    PubMed

    Sun, Chengqun; Gardner, Christina L; Watson, Alan M; Ryman, Kate D; Klimstra, William B

    2014-02-01

    Engineered alphavirus vectors expressing reporters of infection have been used for a number of years due to their relatively low costs for analysis of virus replication and the capacity to utilize imaging systems for longitudinal measurements of growth within single animals. In general, these vectors have been derived from Old World alphaviruses using a second viral subgenomic promoter to express the transgenes, placed either immediately after the nonstructural proteins or at the 3' end of the viral coding sequences. However, the relevance of these vectors to natural infections is questionable, as they have not been rigorously tested for virulence in vivo in comparison with parental viruses or for the retention of the reporter during replication. Here, we report construction of new expression vectors for two Old World arthritogenic alphaviruses (Sindbis and Chikungunya viruses) and two New World encephalitic alphaviruses (eastern and Venezuelan equine encephalitis viruses) based upon either fusion of the reporter protein in frame within nonstructural protein 3 (nsP3) or insertion of the reporter as a cleavable element between the capsid and PE2 structural proteins. We have compared these with a traditional 3' double subgenomic promoter virus expressing either a large, firefly luciferase (fLuc; 1,650 nucleotides), or small, NanoLuc (nLuc; 513 nucleotides), luminescent reporter protein. Results indicate that the nLuc is substantially more stable than fLuc during repeated rounds of infection regardless of the transgene location. However, the capsid-PE2 insertion and nsP3 fusion viruses exhibit the most authentic mimicking of parental virus infection regardless of expressed protein. IMPORTANCE As more antiviral therapeutics and vaccines are developed, rapid and accurate in vivo modeling of their efficacy will be required. However, current alphavirus vectors expressing reporters of infection have not been extensively tested for accurate mimicking of the infection

  17. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice

    PubMed Central

    Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M.; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong

    2016-01-01

    mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes. PMID:26984728

  18. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  19. Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

    PubMed Central

    Bouraï, Mehdi; Lucas-Hourani, Marianne; Gad, Hans Henrik; Drosten, Christian; Jacob, Yves; Tafforeau, Lionel; Cassonnet, Patricia; Jones, Louis M.; Judith, Delphine; Couderc, Thérèse; Lecuit, Marc; André, Patrice; Kümmerer, Beate Mareike; Lotteau, Vincent; Desprès, Philippe; Vidalain, Pierre-Olivier

    2012-01-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus. PMID:22258240

  20. Mapping of Chikungunya virus interactions with host proteins identified nsP2 as a highly connected viral component.

    PubMed

    Bouraï, Mehdi; Lucas-Hourani, Marianne; Gad, Hans Henrik; Drosten, Christian; Jacob, Yves; Tafforeau, Lionel; Cassonnet, Patricia; Jones, Louis M; Judith, Delphine; Couderc, Thérèse; Lecuit, Marc; André, Patrice; Kümmerer, Beate Mareike; Lotteau, Vincent; Desprès, Philippe; Tangy, Frédéric; Vidalain, Pierre-Olivier

    2012-03-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.

  1. The minute virus of canines (MVC) NP1 protein governs the expression of a subset of essential NS proteins via its role in RNA processing.

    PubMed

    Fasina, Olufemi O; Stupps, Stephanie; Figueroa-Cuilan, Wanda; Pintel, David J

    2017-03-29

    Parvoviruses use a variety of means to control the expression of their compact genomes. The Bocaparvovirus Minute Virus of Canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional non-structural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply-spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino terminus of these proteins into the NP1 open reading frame, and splice-site mutations that prevented their expression inhibited virus replication in a host-cell dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing.IMPORTANCE The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the Bocaparvovirus genus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show here that NP1 also controls expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins are together required for virus replication in a host-cell dependent manner.

  2. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

    PubMed Central

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

    2016-01-01

    ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

  3. Bromovirus movement protein genes play a crucial role in host specificity.

    PubMed Central

    Mise, K; Allison, R F; Janda, M; Ahlquist, P

    1993-01-01

    Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small. Images PMID:7682628

  4. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

    PubMed

    Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Yang, Qiwei; Tian, Yufeng; Morales La Madrid, Andres; Mirzoeva, Salida; Bouyer, Patrice G; Xu, David; Walker, Matthew; Cohn, Susan L

    2011-01-01

    Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the

  5. In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

    PubMed Central

    Athmer, Jeremiah; Fehr, Anthony R.; Grunewald, Matthew; Smith, Everett Clinton; Denison, Mark R.

    2017-01-01

    ABSTRACT Coronavirus (CoV) replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER) membranes in replication/transcription complexes (RTC). Many of the CoV nonstructural proteins (nsps) are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV). In MHV, nsp15 contains the genomic RNA packaging signal (P/S), a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA) tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M) protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses. PMID:28143984

  6. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  7. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

    PubMed Central

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

    2016-01-01

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

  8. Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N(pro)) protein of bovine viral diarrhoea virus.

    PubMed

    Mishra, Niranjan; Rajukumar, Katherukamem; Pitale, Shruti Shrikant; Prakash, Anil; Nema, Ram Kumar; Behera, Sthita Pragnya; Dubey, Shiv Chandra

    2010-03-01

    Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N(pro)) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N(pro)-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N(pro) protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N(pro) rabbit serum. When rabbits were immunized with the N(pro) protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N(pro)-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N(pro) antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N(pro) protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.

  9. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

    PubMed

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, C T; Surjit, Milan

    2016-04-26

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

  10. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    SciTech Connect

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.; Borrego, Belen; Brocchi, Emiliana; Armas-Portela, Rosario; Sobrino, Francisco

    2008-05-10

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.

  11. Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

    PubMed Central

    Upadhyay, Anup K.; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J.

    2017-01-01

    The rapid spread of the recent Zika virus (ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 of Zika virus (ZIKV-NS5) is critical for ZIKV replication through the 5′-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space group P21212 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein from Japanese encephalitis virus and suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain. PMID:28291746

  12. Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.

    PubMed

    Heikkinen, Leena S; Kazlauskas, Arunas; Melén, Krister; Wagner, Ralf; Ziegler, Thedi; Julkunen, Ilkka; Saksela, Kalle

    2008-02-29

    NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.

  13. Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

    PubMed Central

    Viskovska, Maria; Anish, Ramakrishnan; Hu, Liya; Chow, Dar-Chone; Hurwitz, Amy M.; Brown, Nicholas G.; Palzkill, Timothy; Estes, Mary K.

    2014-01-01

    ABSTRACT Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated. IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA

  14. In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite

    PubMed Central

    Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

    2015-01-01

    We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed. PMID:26358842

  15. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  16. Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

    PubMed Central

    Copeland, Anna Maria; Altamura, Louis A.; Van Deusen, Nicole M.

    2013-01-01

    Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that