Matrix protein of VSV New Jersey serotype containing methionine to arginine substitutions at positions 48 and 51 allows near-normal host cell gene expression

Abstract

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.

Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system

“…We have inserted the HIV-1 env gene into two different sites in the VSV NJ genome (between the P and M genes and between the G and L genes) and recovered recombinant VSV-HIV-1 gp160 viruses (NP-gp160-MGL and NPMG-gp160-L, respectively) by using the VSV reverse genetics system (Kim & Kang, 2007). We demonstrated that the VSV NJ vector can tolerate a large foreign gene insertion at a position between either the P and M or G and L genes.…”

Expression of HIV-1 gag and env genes using the vesicular stomatitis virus vector system

“…Kim and Kang (2007) found that mutations introduced into the M gene of VSV result in an attenuated virus that exhibits reduced inhibitory effects on host cell gene expression and results in significantly less cytopathic effects within infected cell lines, a finding allowing for the production of avirulent and temperature-sensitive VSV mutants. Most valuable is the existence of VSV in two serotypes; Indiana (VSVInd) and New Jersey (VSVNJ).…”

“…Cells were recovered by centrifugation at 4500 rpm at 4 °C for 10 minutes. according to the manufacturer's specifications in order to recover the recombinant VSVInd(GML)HIV-1gag-env by the VSV recovery protocol (Kim & Kang, 2007). Confluent BHKT7 cells were transfected with 19.4 μg of the plasmid containing VSVInd(GML)HIV-1gag-env and co-transfected with 10 μg of pKS-HP, 10 μg of pKS-HN, and 5 μg of pKS-HL plasmid DNA.…”

Investigation of the Vesicular Stomatitis Virus Matrix Protein: Uncoating and Assembly

“…One could imagine a scenario where this offers VSV an advantage of having different populations of M: (1) a population with the N-terminal amino acids important for membrane binding and budding activity that is used for assembly of progeny virions and (2) a population without those amino acids that cannot be sequestered to the cellular membranes as readily and therefore could interact with NPCs and cause other cytopathic effects. Not only has this been shown for VSV Indiana M protein but it has recently been shown that methionines 48 and 51 of the VSV New Jersey M protein have similar requirements for CPE (104).…”

“…The region on the M protein that is responsible for this interaction has been mapped to amino acids 51-59 with methionine 51 (M51) being the most important for colocalization to NPCs and inhibition of nucleocytoplasmic transport (168). Recently, it was also shown that the VSV New Jersey M48 and M51 residues are important for inhibiting gene expression, suggesting that these may be involved in a similar mechanism for this M protein (104).…”