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Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

ABSTRACT: The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

SUBMITTER: Mitra T

PROVIDER: S-EPMC387666 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

Mitra Tanaji T Sosnovtsev Stanislav V SV Green Kim Y KY

Journal of virology 20040501 9

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA ...[more]

PMID: 15078978

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Project description:To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol II-transcribed RNA in a ribosome-, 5' cap-, and 3' poly(A) tail-independent manner. Moreover, purified glutathione S-transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PP-induced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response.

Dataset Information

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

Publications

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

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