Effect of Vesicular Stomatitis Virus Matrix Protein on Transcription Directed by Host RNA Polymerases I, II, and III

ABSTRACT The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)− RNA synthesis were measured by incorporation of [3H]uridine. v6 andtsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)− RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and α-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.

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Matrix protein of Vesicular stomatitis virus harbours a cryptic mitochondrial-targeting motif

Journal of General Virology ◽

10.1099/vir.0.81762-0 ◽

2006 ◽

Vol 87 (11) ◽

pp. 3379-3384 ◽

Cited By ~ 15

Author(s):

Brian D. Lichty ◽

Heidi McBride ◽

Stephen Hanson ◽

John C. Bell

Keyword(s):

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Protein ◽

Mutational Analysis ◽

M Protein ◽

Mitochondrial Targeting ◽

M Gene ◽

The Matrix ◽

Vesicular Stomatitis ◽

Green Fluorescent

Vesicular stomatitis virus (VSV) is a rhabdovirus that has attracted attention of late as an oncolytic virus and as a vaccine vector. Mutations in the matrix (M) gene of VSV yield attenuated strains that may be very useful in both settings. As a result of this interest in the M protein, this study analysed various M–green fluorescent protein (GFP) fusion constructs. Remarkably, fusion of the N terminus of the M protein to GFP targeted the fluorescent protein to the surface of mitochondria. Mutational analysis indicated that a mitochondrial-targeting motif exists within aa 33–67. Expression of these fusion proteins led to loss of mitochondrial membrane permeability and to an alteration in mitochondrial organization mirroring that seen during viral infection. In addition, a portion of the M protein present in infected cells co-purified with mitochondria. This work may indicate a novel function for this multifunctional viral protein.

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Modifications of the PSAP region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo

Journal of General Virology ◽

10.1099/vir.0.83096-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2559-2567 ◽

Cited By ~ 17

Author(s):

Takashi Irie ◽

Elena Carnero ◽

Atsushi Okumura ◽

Adolfo García-Sastre ◽

Ronald N. Harty

Keyword(s):

Cell Culture ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Virus Assembly ◽

Host Protein ◽

M Protein ◽

Recombinant Viruses ◽

Enhanced Production ◽

The Matrix ◽

Vesicular Stomatitis

The matrix (M) protein of vesicular stomatitis virus (VSV) is a multi-functional protein involved in virus assembly, budding and pathogenesis. The 24PPPY27 late (L) domain of the M protein plays a key role in virus budding, whereas amino acids downstream of the PPPY motif contribute to host protein shut-off and pathogenesis. Using a panel of 37PSAP40 recombinant viruses, it has been demonstrated previously that the PSAP region of M does not possess L-domain activity similar to that of PPPY in BHK-21 cells. This study reports the unanticipated finding that these PSAP recombinants were attenuated in cell culture and in mice compared with control viruses. Indeed, PSAP recombinant viruses exhibited a small-plaque phenotype, reduced CPE, reduced levels of activated caspase-3, enhanced production of IFN-β and reduced titres in the lungs and brains of infected mice. In particular, recombinant virus M6PY>A4-R34E was the most severely attenuated, exhibiting little or no CPE in cell culture and undetectable titres in the lungs and brains of infected mice. These findings indicate an important role for the PSAP region (aa 33–44) of the M protein in the pathology of VSV infection and may have implications for the development of VSV as a vaccine and/or oncolytic vector.

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Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy

RSC Advances ◽

10.1039/c7ra13656k ◽

2018 ◽

Vol 8 (22) ◽

pp. 12104-12115 ◽

Cited By ~ 2

Author(s):

Ke Men ◽

Rui Zhang ◽

Xueyan Zhang ◽

Rong Huang ◽

Guonian Zhu ◽

...

Keyword(s):

Gene Therapy ◽

Colon Cancer ◽

Colon Carcinoma ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Cancer Gene Therapy ◽

Viral Gene ◽

Cancer Gene ◽

Viral Gene Therapy ◽

Vesicular Stomatitis

Liposome–protamine complex delivered VSVMP mRNA efficiently inhibits C26 colon carcinoma with safety, providing an alternative strategy for non-viral gene therapy.

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Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology

Journal of Virology ◽

10.1128/jvi.76.16.8011-8018.2002 ◽

2002 ◽

Vol 76 (16) ◽

pp. 8011-8018 ◽

Cited By ~ 53

Author(s):

Himangi R. Jayakar ◽

Michael A. Whitt

Keyword(s):

Amino Acids ◽

Vesicular Stomatitis Virus ◽

Recombinant Virus ◽

M Protein ◽

M Gene ◽

Cell Rounding ◽

Matrix Gene ◽

The Matrix ◽

Infected Cells ◽

Vesicular Stomatitis

ABSTRACT 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 of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene.

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Phenotypes of vesicular stomatitis virus mutants with mutations in the PSAP motif of the matrix protein

Journal of General Virology ◽

10.1099/vir.0.039800-0 ◽

2012 ◽

Vol 93 (4) ◽

pp. 857-865 ◽

Cited By ~ 16

Author(s):

Linda Obiang ◽

Hélène Raux ◽

Malika Ouldali ◽

Danielle Blondel ◽

Yves Gaudin

Keyword(s):

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Virus Assembly ◽

Susceptibility Gene ◽

Point Mutations ◽

Binding Motif ◽

Single Mutation ◽

Amino Terminal ◽

Vesicular Stomatitis ◽

Late Domains

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

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Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

Journal of Virology ◽

10.1128/jvi.01668-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2611-2622 ◽

Cited By ~ 42

Author(s):

Subash C. Das ◽

Debasis Panda ◽

Debasis Nayak ◽

Asit K. Pattnaik

Keyword(s):

Plasma Membrane ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Protein ◽

Dynamic Imaging ◽

Viral Envelope ◽

M Protein ◽

Recombinant Viruses ◽

Infected Cells ◽

Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

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Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein

Journal of Virology ◽

10.1128/jvi.00955-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12139-12150 ◽

Cited By ~ 22

Author(s):

Chad E. Mire ◽

Derek Dube ◽

Sue E. Delos ◽

Judith M. White ◽

Michael A. Whitt

Keyword(s):

G Protein ◽

Vesicular Stomatitis Virus ◽

Conformational Changes ◽

Matrix Protein ◽

Low Ph ◽

M Protein ◽

Steady Increase ◽

Protein Dissociation ◽

Vesicular Stomatitis

ABSTRACT To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating.

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Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release

Journal of Virology ◽

10.1128/jvi.74.21.9818-9827.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 9818-9827 ◽

Cited By ~ 123

Author(s):

Himangi R. Jayakar ◽

K. Gopal Murti ◽

Michael A. Whitt

Keyword(s):

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Electron Microscopic Examination ◽

M Protein ◽

Virus Yield ◽

Virus Budding ◽

Electron Microscopic ◽

Gag Proteins ◽

Virion Release ◽

Vesicular Stomatitis

ABSTRACT The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.

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Role of Residues 121 to 124 of Vesicular Stomatitis Virus Matrix Protein in Virus Assembly and Virus-Host Interaction

Journal of Virology ◽

10.1128/jvi.80.8.3701-3711.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3701-3711 ◽

Cited By ~ 16

Author(s):

John H. Connor ◽

Margie O. McKenzie ◽

Douglas S. Lyles

Keyword(s):

Vesicular Stomatitis Virus ◽

Viral Protein ◽

Virus Assembly ◽

Mrna Translation ◽

Mutant Virus ◽

M Protein ◽

Viral Mrna ◽

Wild Type ◽

Vesicular Stomatitis ◽

Self Association

ABSTRACT The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on virus assembly, the mutant virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant virus produced more viral mRNA, showing that the mutant virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in virus assembly but is involved in virus-host interactions and VSV replication by augmenting viral-mRNA translation.

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