The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Richard M Elliott - One of the best experts on this subject based on the ideXlab platform.
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Evolution of the Bunyamwera Virus Polymerase To Accommodate Deletions within Genomic Untranslated Region Sequences
2016Co-Authors: Béryl Mazel-sanchez, Richard M ElliottAbstract:The untranslated regions (UTR) present at the ends of bunyaVirus genome segments are required for essential steps in the Virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replica-tion as well as for mRNA transcription termination. For the prototype bunyaVirus, Bunyamwera Virus (BUNV), only the termi-nal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different Viruses are highly variable. We previously generated re-combinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these Viruses, the Viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant Viruses, with the vast majority of the amino acid changes occurring in the C-termi-nal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a Virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensi-tive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae
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Evolution of the Bunyamwera Virus Polymerase To Accommodate Deletions within Genomic Untranslated Region Sequences
Journal of virology, 2015Co-Authors: Béryl Mazel-sanchez, Richard M ElliottAbstract:The untranslated regions (UTR) present at the ends of bunyaVirus genome segments are required for essential steps in the Virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replication as well as for mRNA transcription termination. For the prototype bunyaVirus, Bunyamwera Virus (BUNV), only the terminal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different Viruses are highly variable. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these Viruses, the Viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant Viruses, with the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a Virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae. IMPORTANCE Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera Viruses by deleting sequences within both the 3′ and 5′ untranslated regions (UTR) on each genome segment and showed that the Viruses regained fitness following serial passage in cell culture. The fitter Viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognizing the mutant UTRs. We suggest that deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene encoding the NSs protein and introduction of temperature-sensitive mutations, in the design of attenuated bunyaViruses that could have potential as vaccines.
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Role of Bunyamwera OrthobunyaVirus NSs Protein in Infection of Mosquito Cells
PLoS Neglected Tropical Diseases, 2012Co-Authors: Agnieszka Szemiel, Anna-bella Failloux, Richard M ElliottAbstract:BACKGROUND: Bunyamwera orthobunyaVirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera Virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. METHODOLOGY AND PRINCIPAL FINDINGS: To determine potential functions of the NSs protein in mosquito cells, replication of wild-type Virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and Virus yields at various times post infection. Both Viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant Viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced Virus replication in U4.4 cells transfected with Virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion Virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type Virus, infection rates by the mutant Virus were lower and more variable. If the NSs deletion Virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type Virus. CONCLUSIONS/SIGNIFICANCE: Bunyamwera Virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.
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the n terminus of Bunyamwera orthobunyaVirus nss protein is essential for interferon antagonism
Journal of General Virology, 2010Co-Authors: Ingeborg Van Knippenberg, Charles Carltonsmith, Richard M ElliottAbstract:Bunyamwera Virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera Virus (rBUNdelNSs2) was identified and characterized. This mutant Virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera Virus to counteract host antiviral responses.
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the unique architecture of Bunyamwera Virus factories around the golgi complex
Cellular Microbiology, 2008Co-Authors: Juan Fontana, Richard M Elliott, Noelia Lopezmontero, Josejesus Fernandez, Cristina RiscoAbstract:Viral factories are novel structures built by Viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyaVirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. Numerous ribonucleoproteins were released from purified tubes disrupted in vitro. Actin and myosin I were identified by peptide mass fingerprinting in isolated tubes while actin and the viral NSm non-structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. Studies with NSm deletion mutants and drugs affecting actin showed that both NSm and actin are key factors for tube and Virus assembly in Golgi. Three-dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to Golgi stacks and make contacts with intracellular Viruses. We propose that this new structure, unique among enveloped Viruses, assembles in association with the most stable component of Golgi stacks, the actin-containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories.
Friedemann Weber - One of the best experts on this subject based on the ideXlab platform.
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Efficient cDNA-based rescue of La Crosse bunyaViruses expressing or lacking the nonstructural proteinNSs
2016Co-Authors: Gjon Blakqori, Friedemann WeberAbstract:La Crosse Virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera Virus (Lowen et al., Virology 330:493–500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3 ends trimmed by the hepatitis delta Virus ribozyme. As has been shown for Bunyamwera Virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 108 infectious Viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type Virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutof
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Bunyamwera Virus nonstructural protein nss counteracts interferon regulatory factor 3 mediated induction of early cell death
Journal of Virology, 2003Co-Authors: Alain Kohl, Reginald F Clayton, Friedemann Weber, Anne Bridgen, R E Randall, Richard M ElliottAbstract:The genome of Bunyamwera Virus (BUN; family Bunyaviridae, genus OrthobunyaVirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type Virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.
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Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs
Journal of virology, 2003Co-Authors: Hein Streitenfeld, John K. Fazakerley, Anne Bridgen, Richard M Elliott, Amanda Boyd, Friedemann WeberAbstract:Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera Virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant Virus that does not express NSs (BUNdelNSs Virus). Furthermore, the sensitivity of Viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaViruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
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Bunyamwera bunyaVirus nonstructural protein nss counteracts the induction of alpha beta interferon
Journal of Virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
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Bunyamwera BunyaVirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon
Journal of virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
Anne Bridgen - One of the best experts on this subject based on the ideXlab platform.
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Bunyamwera Virus nonstructural protein nss counteracts interferon regulatory factor 3 mediated induction of early cell death
Journal of Virology, 2003Co-Authors: Alain Kohl, Reginald F Clayton, Friedemann Weber, Anne Bridgen, R E Randall, Richard M ElliottAbstract:The genome of Bunyamwera Virus (BUN; family Bunyaviridae, genus OrthobunyaVirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type Virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.
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Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs
Journal of virology, 2003Co-Authors: Hein Streitenfeld, John K. Fazakerley, Anne Bridgen, Richard M Elliott, Amanda Boyd, Friedemann WeberAbstract:Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera Virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant Virus that does not express NSs (BUNdelNSs Virus). Furthermore, the sensitivity of Viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaViruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
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Effects of a point mutation in the 3 ' end of the S genome segment of naturally occurring and engineered Bunyamwera Viruses
Journal of General Virology, 2003Co-Authors: Alain Kohl, Anne Bridgen, John N Barr, Ewan F Dunn, Richard M ElliottAbstract:The genome of Bunyamwera Virus (BUN) consists of three segments of single-stranded RNA of negative polarity. The smallest segment, S, encodes the N protein and a nonstructural protein called NSs. We recently described a mutant Virus (BUNdelNSs) that does not express NSs but overexpresses N and grows to lower titres than wild-type (wt) BUN. Here we report a BUNdelNSs variant that expresses lower levels of N protein and grows to higher titres. Sequencing of the 3′ and 5′ termini of the BUNdelNSs S RNA segment and analysis using a minireplicon system show that the N overexpressing phenotype results from a single nucleotide substitution at position 16 in the 3′ terminus. This mutation could also be detected in wtBUN populations, and was isolated by plaquing a ‘wt’ variant carrying the mutation. This variant was found to express increased N and NSs levels, and grew to lower titres than wtBUN.
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Bunyamwera bunyaVirus nonstructural protein nss counteracts the induction of alpha beta interferon
Journal of Virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
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Bunyamwera BunyaVirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon
Journal of virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
John N Barr - One of the best experts on this subject based on the ideXlab platform.
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Amino acid changes within the Bunyamwera Virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates.
Journal of General Virology, 2010Co-Authors: Cheryl T. Walter, Diana Filipa Costa Bento, Ana Guerrero Alonso, John N BarrAbstract:The genome of Bunyamwera Virus (BUNV) comprises three RNA segments that are encapsidated by the Virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the Virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation.
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Bunyamwera Virus can repair both insertions and deletions during RNA replication.
RNA (New York N.Y.), 2010Co-Authors: Cheryl T. Walter, John N BarrAbstract:The genomic termini of RNA Viruses contain essential cis-acting signals for such diverse functions as packaging, genome translation, mRNA transcription, and RNA replication, and thus preservation of their sequence integrity is critical for Virus viability. Sequence alteration can arise due to cellular mechanisms that add or remove nucleotides from terminal regions, or, alternatively, from introduction of sequence errors through nucleotide misincorporation by the error-prone viral RNA-dependent RNA polymerase (RdRp). To preserve template function, many RNA Viruses utilize repair mechanisms to prevent accumulation of terminal alterations. Here we show that Bunyamwera Virus (BUNV), the prototype of the Bunyaviridae family of segmented negative-sense RNA Viruses, also can repair its genomic termini. When an intact nontranslated region (NTR) was added to the anti-genomic 3' end, it was precisely removed, to restore both length and RNA synthesis function of the wild-type template. Furthermore, when nucleotides were removed from the anti-genome 3' end, and replaced with a duplicate and intact NTR, both the external NTR were removed, and the missing nucleotides were restored, thus, indicating that the BUNV RdRp can both remove and add nucleotides to the template. We show that the mechanism for repair of terminal extensions is likely that of internal entry of the viral RdRp during genome synthesis. Possible mechanisms for repair of terminal deletions are discussed.
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Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera Virus.
RNA (New York N.Y.), 2009Co-Authors: Bjorn-patrick Mohl, John N BarrAbstract:Bunyamwera Virus (BUNV) is the prototypic member of both the OrthobunyaVirus genus and the Bunyaviridae family of negative stranded RNA Viruses. In common with all negative stranded RNA Viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the Virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA Virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyaVirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffraction data. Here we extend this work to show that this soluble N protein is able to oligomerize and bind RNA to form a highly uniform RNP complex, which exhibits characteristics in common with the viral RNP. By extracting and sequencing RNAs bound to these model RNPs, we determined the stoichiometry of N-RNA association to be ;12 nucleotides per N monomer. In addition, we defined the minimal sequence requirement for BUNV RNA replication. By comparing this minimal sequence to those bound to our model RNP, we conclude that N protein does not obligatorily require a sequence or structure for RNA encapsidation.
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Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera Virus.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: John W Rodgers, Qingxian Zhou, Todd J Green, John N Barr, Ming LuoAbstract:Bunyamwera Virus (BUNV) is the prototypic member of the Bunyaviridae family of segmented negative-sense RNA Viruses. The BUNV nucleocapsid protein has been cloned and expressed in Escherichia coli. The purified protein has been crystallized and a complete data set has been collected to 3.3 angstroms resolution at a synchrotron source. Crystals of the nucleocapsid protein belong to space group C2, with unit-cell parameters a = 384.7, b = 89.8, c = 89.2 angstroms, beta = 94.4 degrees . Self-rotation function analysis of the X-ray diffraction data has provided insight into the oligomeric state of the protein as well as the orientation of the oligomers in the asymmetric unit. The structure determination of the protein is ongoing.
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Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera Virus.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: John W Rodgers, Qingxian Zhou, John N Barr, Todd Green, Ming LuoAbstract:Bunyamwera Virus (BUNV) is the prototypic member of the Bunyaviridae family of segmented negative-sense RNA Viruses. The BUNV nucleocapsid protein has been cloned and expressed in Escherichia coli. The purified protein has been crystallized and a complete data set has been collected to 3.3 A resolution at a synchrotron source. Crystals of the nucleocapsid protein belong to space group C2, with unit-cell parameters a = 384.7, b = 89.8, c = 89.2 A, β = 94.4°. Self-rotation function analysis of the X-ray diffraction data has provided insight into the oligomeric state of the protein as well as the orientation of the oligomers in the asymmetric unit. The structure determination of the protein is ongoing.
John K. Fazakerley - One of the best experts on this subject based on the ideXlab platform.
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Host Inflammatory Response to Mosquito Bites Enhances the Severity of ArboVirus Infection
Immunity, 2016Co-Authors: Marieke Pingen, Steven R. Bryden, Emilie Pondeville, Andres Merits, Esther Schnettler, John K. Fazakerley, Alain Kohl, Gerard J. Graham, Clive S MckimmieAbstract:Aedes aegypti mosquitoes are responsible for transmitting many medically important Viruses such as those that cause Zika and dengue. The inoculation of Viruses into mosquito bite sites is an important and common stage of all mosquito-borne Virus infections. We show, using Semliki Forest Virus and Bunyamwera Virus, that these Viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained Virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated Virus infection by encouraging the entry and infection of Virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of Virus infection.
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Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs
Journal of virology, 2003Co-Authors: Hein Streitenfeld, John K. Fazakerley, Anne Bridgen, Richard M Elliott, Amanda Boyd, Friedemann WeberAbstract:Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera Virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant Virus that does not express NSs (BUNdelNSs Virus). Furthermore, the sensitivity of Viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaViruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
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Bunyamwera bunyaVirus nonstructural protein nss counteracts the induction of alpha beta interferon
Journal of Virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
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Bunyamwera BunyaVirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon
Journal of virology, 2002Co-Authors: Friedemann Weber, John K. Fazakerley, Anne Bridgen, R E Randall, Hein Streitenfeld, Nina Kessler, Richard M ElliottAbstract:Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many Viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera Virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant Virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera Virus.
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Bunyamwera bunyaVirus nonstructural protein nss is a nonessential gene product that contributes to viral pathogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Anne Bridgen, John K. Fazakerley, Friedemann Weber, Richard M ElliottAbstract:Abstract Bunyamwera Virus (family Bunyaviridae, genus BunyaVirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant Virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) Virus, BUNdelNSs exhibited a smaller plaque size and generated titers of Virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt Virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of Virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera Virus from a noninducer to an inducer of an interferon-β promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyaVirus NSs protein has several functions in the Virus life cycle and contributes to viral pathogenesis.
