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Grant Mcfadden - One of the best experts on this subject based on the ideXlab platform.
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mutations in active site residues of the uracil dna glycosylase encoded by vaccinia Virus are incompatible with Virus Viability
Journal of Virology, 1996Co-Authors: Kimberly S Ellison, Wei Peng, Grant McfaddenAbstract:The D4R gene of vaccinia Virus encodes a functional uracil-DNA glycosylase that is essential for viral Viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia Virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia Virus DNA replication. Three active-site amino acids of the vaccinia Virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia Virus ts4149-infected cells in order to assess the recombination events that allowed Virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only Viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia Virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.
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myxoma Virus and shope fibroma Virus encode dual specificity tyrosine serine phosphatases which are essential for Virus Viability
Virology, 1995Co-Authors: Karen L Mossman, Chris Upton, Hanne L Ostergaard, Grant McfaddenAbstract:Abstract Sequence analysis of the genomes of the LeporipoxViruses myxoma Virus and Shope fibroma Virus (SFV) led to thediscovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma Bam HI-I fragment and the SFV Bam HI-M fragment, and the resulting encoded proteins were designated l1L and M1L, respectively. The localization and orientation of the myxoma l1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the OrthopoxViruses vaccinia Virus and variola Virus. The myxoma l1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro . To assess whether this is a common feature of poxVirus phosphatases, myxoma 11 L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma l1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type l1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma l1L phosphatase, we constructed a recombinant myxoma Virus containing a disrupted l1L open reading frame. This l1L mutant Virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure Virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma l1L dual specificity phosphatase is an essential factor for Virus Viability.
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A poxVirus-encoded uracil DNA glycosylase is essential for Virus Viability.
Journal of Virology, 1993Co-Authors: David T. Stuart, Edward G Niles, Meghan A. Higman, Chris Upton, Grant McfaddenAbstract:Infection ofcultured mammaliancells withtheLeporipoxVirus Shopefibroma Virus(SFV)causes the induction ofanovel uracil DNA glycosylase activity inthecytoplasms oftheinfected cells. Theinduction ofthis activity, early ininfection, correlates withtheearly expression oftheSFVBamHID6R open reading frame whichpossessessignificant protein sequencesimilarity toeukaryotic andprokaryotic uracil DNA glycosylases. TheSFVBamHID6R open reading frameandthehomologous Hindlll D4R open reading framefromthe OrthopoxVirus vaccinia Virus were cloned undertheregulation ofa phageT7 promoter andexpressed in Escherichia coli as insoluble high-molecular-weight aggregates. Duringelectrophoresis on sodiumdodecyl sulfate-polyacrylamide gels, theE.coli-expressed proteins migrate withan apparent molecular mass of25kDa. Theinsoluble protein aggregate generated byexpression inE.coli was solubilized inurea and,following a subsequent refolding step, displayed theability toexcise uracil residues fromdouble-stranded plasmid DNA substrates, withthesubsequent formation ofapyrimidinic sites. Theviral enzyme,like allother characterized uracil DNA glycosylases, isactive inthepresenceofhighconcentrations ofEDTA,issubstrate inhibited by uracil, anddoesnotdisplay anyendonuclease activity. Attempts toinactivate theHindIII D4Rgeneofvaccinia Virus bytargeted insertion ofa dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion ofa frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described forherpesViruses, thepoxVirus enzyme isessential forVirus Viability.
Zuzhang Wei - One of the best experts on this subject based on the ideXlab platform.
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cysteine residues of the porcine reproductive and respiratory syndrome Virus orf5a protein are not essential for Virus Viability
Virus Research, 2015Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Hongjie Fan, Zuzhang Wei, Yan Zhou, Shishan Yuan, Guangzhi TongAbstract:ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome Virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on Virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for Virus Viability, but all serine mutant clones produced infectious progeny Viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.
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porcine reproductive and respiratory syndrome Virus orf5a protein is essential for Virus Viability
Virus Research, 2013Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Tao Lin, Ting Huang, Huochun Yao, Guangzhi Tong, Hongjie Fan, Zuzhang WeiAbstract:Abstract It has been shown that ORF5a protein in EAV is important but not essential for Virus infectivity. In this study, we found that RNA changes in the overlapping region (1–104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for Virus Viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious Virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious Virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for Virus Viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.
Guangzhi Tong - One of the best experts on this subject based on the ideXlab platform.
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cysteine residues of the porcine reproductive and respiratory syndrome Virus orf5a protein are not essential for Virus Viability
Virus Research, 2015Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Hongjie Fan, Zuzhang Wei, Yan Zhou, Shishan Yuan, Guangzhi TongAbstract:ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome Virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on Virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for Virus Viability, but all serine mutant clones produced infectious progeny Viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.
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porcine reproductive and respiratory syndrome Virus orf5a protein is essential for Virus Viability
Virus Research, 2013Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Tao Lin, Ting Huang, Huochun Yao, Guangzhi Tong, Hongjie Fan, Zuzhang WeiAbstract:Abstract It has been shown that ORF5a protein in EAV is important but not essential for Virus infectivity. In this study, we found that RNA changes in the overlapping region (1–104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for Virus Viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious Virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious Virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for Virus Viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.
Moisés Gómez Mateu - One of the best experts on this subject based on the ideXlab platform.
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Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease Virus
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Miguel Angel Martínez, Núria Verdaguer, Moisés Gómez MateuAbstract:AphthoViruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for Virus Viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the Virus harboring it. Foot-and-mouth disease Virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental Virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that Viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA Viruses in nature.
Lichang Sun - One of the best experts on this subject based on the ideXlab platform.
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cysteine residues of the porcine reproductive and respiratory syndrome Virus orf5a protein are not essential for Virus Viability
Virus Research, 2015Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Hongjie Fan, Zuzhang Wei, Yan Zhou, Shishan Yuan, Guangzhi TongAbstract:ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome Virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on Virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for Virus Viability, but all serine mutant clones produced infectious progeny Viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.
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porcine reproductive and respiratory syndrome Virus orf5a protein is essential for Virus Viability
Virus Research, 2013Co-Authors: Lichang Sun, Runxia Liu, Xiaomin Wang, Fei Gao, Tao Lin, Ting Huang, Huochun Yao, Guangzhi Tong, Hongjie Fan, Zuzhang WeiAbstract:Abstract It has been shown that ORF5a protein in EAV is important but not essential for Virus infectivity. In this study, we found that RNA changes in the overlapping region (1–104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for Virus Viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious Virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious Virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for Virus Viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.
