Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression

Abstract

In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed 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.

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors

“…Results presented in this study indicate that VSV with RABV-G or LCMV-G spreads primarily among synaptically connected neurons. To conduct physiological analysis of putative connected pairs, it was necessary to use a virus with a mutant form of the M gene for these experiments (M51R mutant) (25,26). Because the M protein shuts off host transcription and RNA export, M is thought to be the main component of G-deleted VSV's rapid cytotoxicity (26).…”

“…Additionally, the authors note that on page 19245, left column, third full paragraph, lines 1-6, "WA09 (WiCell), RUES1, RUES2 (A. Brivanlou, The Rockefeller University, New York) hESCs, and the hiPSC lines Fib2-iPS4 and Fib2-iPS5 (George Daley, Children's Hospital, Boston) were cultured on Geltrex-coated plates (Invitrogen) in chemically defined media containing Heregulin β (10 ng/mL), Activin A (10 ng/mL), LR-Igf (200 ng/ mL), and Fgf2 (8 ng/mL) as described previously (24)" should instead appear as "WA09 (WiCell), RUES1, RUES2 (A. Brivanlou, The Rockefeller University, New York) hESCs, and the hiPSC lines Fib2-iPS4 and Fib2-iPS5 (George Daley, Children's Hospital, Boston) were cultured on Geltrex-coated plates (Invitrogen) in chemically defined media containing Heregulin β (10 ng/mL), Activin A (10 ng/mL), LR-Igf (200 ng/mL), and Fgf2 (8 ng/mL) as described previously (25)." Contributed by Constance L. Cepko, July 7, 2011 (sent for review June 10, 2011) To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit.…”

“…5 G-K). A VSV with a point mutation in the M gene (M51R) (25,26) was used for these experiments because this variant had been shown to prolong neuronal survival in hippocampal cultures (25). The cultures that were transfected with LCMV-G and ChR2 were used for this experiment, to allow light to be used to stimulate the transfected/infected neuron because of the presence of ChR2 (Fig.…”

Cytopathogenesis and Inhibition of Host Gene Expression by RNA Viruses

“…This mutation results in substitution of arginine for the methionine at position 51 (M51R mutation) of the 229-aa M protein. The M51R mutant M protein is 10-to 100-fold less active than is the wild-type M protein in the inhibition of host-directed transcription in cotransfection experiments (3,10,39). An additional M protein mutation, which results in a two-to three-fold reduction in inhibitory activity, has been identified (N163D) in viruses isolated from persistently infected cells (3).…”

Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity

“…In addition, mutations in the M gene were detected frequently in persistently VSV-infected cells and some of these mutations (e.g. M51R) were shown to abolish the M protein-mediated host shut-off (Ahmed & Lyles, 1997;Desforges et al, 2001). Correspondingly, these mutants do not suppress IFN synthesis and have been proposed as oncolytic viruses for the therapy of tumours that show defects in the type I IFN system (Barber, 2004(Barber, , 2005Lichty et al, 2004;Stojdl et al, 2000;Wollmann et al, 2007).…”