Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression (original) (raw)
In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits hostdirected gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression in cells persistently infected with VSV. Viruses isolated from L cells persistently infected with VSV inhibited host RNA synthesis more slowly than wild-type (wt) VSV. M genes of the persistent viral population were cloned and sequenced. One mutation, an N to D change at position 163 of the protein sequence (N163D), was common to all the molecular clones. The N163D M protein was synthesized from transfected mRNA at a rate that was 30% of that of wt M protein, but was turned over at a rate that was similar to that of wt M protein. Transfection of mRNA encoding N163D M protein inhibited expression of a cotransfected target gene encoding chloramphenicol acetyl transferase (CAT), but the inhibition was 6 to 10 times less effective than transfection of equivalent amounts of wt M mRNA. This difference could not be accounted for by differences in translation of CAT mRNA. Thus, when the differences in M protein expression were taken into account, N163D M protein was 2 to 3 times less effective than wt M protein in the inhibition of host-directed gene expression, similar to the differences in host transcription observed in virus-infected cells. Point mutations in addition to the N163D mutation were found in about half of the M gene molecular clones. The M gene of an independently isolated molecular clone, N163D.2, contained two additional point mutations in its carboxy terminal region. N163D.2 M protein was highly defective in inhibition of host gene expression and was turned over more rapidly than wt M protein. These results support the idea that M gene mutations contribute to a reduced cytopathic effect in cells persistently infected with VSV.
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Virology, 1996
The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts of in vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700-11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature. ᭧
Journal of Virology
A new transcription unit was generated in the 3' noncoding region of the vesicular stomatitis virus (VSV) glycoprotein gene by introducing the smallest conserved sequence found at each VSV gene junction. This sequence was introduced into a DNA copy of the VSV genome from which infectious VSV can be derived. It contained an 11-nucleotide putative transcription stop/polyadenylation signal for the glycoprotein mRNA, an intergenic dinucleotide, and a 10-nucleotide putative transcription start sequence preceding a downstream foreign gene encoding the bacterial enzyme chloramphenicol acetyltransferase. Infectious recombinant VSV was recovered from this construct and was found to express high levels of functional chloramphenicol acetyltransferase mRNA and protein. The recombinant virus grew to wild-type titers of 5 x 10(9)/ml, and expression of the foreign gene was completely stable for at least 15 passages involving 10(6)-fold expansion at each passage. These results define functional...
Impact of Vesicular Stomatitis Virus M Proteins on Different Cellular Functions
PLOS ONE, 2015
Three different matrix (M) proteins termed M1, M2 and M3 have been described in cells infected with vesicular stomatitis virus (VSV). Individual expression of VSV M proteins induces an evident cytopathic effect including cell rounding and detachment, in addition to a partial inhibition of cellular protein synthesis, likely mediated by an indirect mechanism. Analogous to viroporins, M1 promotes the budding of new virus particles; however, this process does not produce an increase in plasma membrane permeability. In contrast to M1, M2 and M3 neither interact with the cellular membrane nor promote the budding of double membrane vesicles at the cell surface. Nonetheless, all three species of M protein interfere with the transport of cellular mRNAs from the nucleus to the cytoplasm and also modulate the redistribution of the splicing factor. The present findings indicate that all three VSV M proteins share some activities that interfere with host cell functions.
Journal of Virology, 2002
The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression...
Journal of General Virology, 1986
A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15 ~ difference at the nucleotide level and 10Y/oo difference at the amino acid level; the majority of the changes were located in the Y half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50~ nucleotide and 33 ~ amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity. 0000-7046 © 1986 SGM Downloaded from www.microbiologyresearch.org by IP: 54.157.22.242 On: Fri, 26 Aug 2016 09:24:38 1352 B. P. RAE AND R. M. ELLIOTT
Virology, 2007
The matrix (M) protein of vesicular stomatitis virus (VSV) plays significant roles in the replication of VSV through its involvement in the assembly of virus particles as well as by facilitating the evasion of innate host cell defense mechanisms. The presence of methionine at position 51 (M51) of the matrix (M) protein of the VSV Indiana serotype (VSV Ind ) has been proven to be crucial for cell rounding and inhibition of host cell gene expression. The M protein of VSV Ind with the substitution of M51 with arginine (R:M51R) results in the loss of inhibitory effects on host cell gene expression. The VSV Ind expressing the M(M51R) protein became the attractive oncolytic virus which is safer and more tumor-specific because the normal cells can clear the mutant VSV Ind easily but tumor cells are susceptible to the virus because a variety of tumor cells lack innate antiviral activities. We have studied the role of the methionines at positions 48 and 51 of the M protein of the New Jersey serotype of VSV (VSV NJ ) in the induction of cytopathic effects (CPE) and host cell gene expression. We have generated human embryonic kidney 293 cell lines inducibly expressing M proteins with M to R mutations at positions 48 and 51, either separately or together as a double mutant, and examined expression of heat shock protein 70 (HSP70) as an indicator of host cell gene expression. We have also generated recombinant VSV NJ encoding the mutant M proteins M(M48R) or M(M48R + M51R) for the first time and tested for the expression of HSP70 in infected cells. Our results demonstrated that the M51 of VSV NJ M proteins has a major role in cell rounding and in suppressing the host cell gene expression either when the M protein was expressed alone in inducible cell lines or when expressed together with other VSV proteins by the recombinant VSV NJ . Amino acid residue M48 may also have some role in cell rounding and in the inhibitory effects of VSV NJ M, which was demonstrated by the fact that the cell line expressing the double substitution mutant M(M48R + M51R) exhibited the least cytopathic effects and the least inhibitory effect on host cell gene expression.
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