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

2006 ◽  
Vol 80 (8) ◽  
pp. 3701-3711 ◽  
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.

scholarly journals Immune Response in the Absence of Neurovirulence in Mice Infected with M Protein Mutant Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (18) ◽  
pp. 9273-9277 ◽  
Author(s):  
Maryam Ahmed ◽  
Tracie R. Marino ◽  
Shelby Puckett ◽  
Nancy D. Kock ◽  
Douglas S. Lyles
Keyword(s):  
Central Nervous System ◽  
Immune Response ◽  
Nervous System ◽  
Vesicular Stomatitis Virus ◽  
M Protein ◽  
Wild Type ◽  
Protein Mutants ◽  
The Central Nervous System ◽  
Vesicular Stomatitis

ABSTRACT Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.

scholarly journals The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly

2000 ◽  
Vol 74 (5) ◽  
pp. 2239-2246 ◽  
Author(s):  
Clinton S. Robison ◽  
Michael A. Whitt
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Virus Release ◽  
Wild Type ◽  
Virus Budding ◽  
Recombinant Viruses ◽  
Vesicular Stomatitis ◽  
High Level

ABSTRACT In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.

scholarly journals Functional Analysis of Late-Budding Domain Activity Associated with the PSAP Motif within the Vesicular Stomatitis Virus M Protein

2004 ◽  
Vol 78 (14) ◽  
pp. 7823-7827 ◽  
Author(s):  
Takashi Irie ◽  
Jillian M. Licata ◽  
Himangi R. Jayakar ◽  
Michael A. Whitt ◽  
Peter Bell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Reverse Genetics ◽  
M Protein ◽  
Plaque Size ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Flanking Sequences ◽  
Vesicular Stomatitis

ABSTRACT A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.

scholarly journals The Matrix Protein of Vesicular Stomatitis Virus Binds Dynamin for Efficient Viral Assembly

2010 ◽  
Vol 84 (24) ◽  
pp. 12609-12618 ◽  
Author(s):  
Hélène Raux ◽  
Linda Obiang ◽  
Nicolas Richard ◽  
Francis Harper ◽  
Danielle Blondel ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Matrix Protein ◽  
Mutant Virus ◽  
Endocytic Pathway ◽  
M Protein ◽  
Pleckstrin Homology Domain ◽  
Single Mutation ◽  
Amino Terminal ◽  
Vesicular Stomatitis

ABSTRACT Matrix proteins (M) direct the process of assembly and budding of viruses belonging to the Mononegavirales order. Using the two-hybrid system, the amino-terminal part of vesicular stomatitis virus (VSV) M was shown to interact with dynamin pleckstrin homology domain. This interaction was confirmed by coimmunoprecipitation of both proteins in cells transfected by a plasmid encoding a c-myc-tagged dynamin and infected by VSV. A role for dynamin in the viral cycle (in addition to its role in virion endocytosis) was suggested by the fact that a late stage of the viral cycle was sensitive to dynasore. By alanine scanning, we identified a single mutation of M protein that abolished this interaction and reduced virus yield. The adaptation of mutant virus (M.L4A) occurred rapidly, allowing the isolation of revertants, among which the M protein, despite having an amino acid sequence distinct from that of the wild type, recovered a significant level of interaction with dynamin. This proved that the mutant phenotype was due to the loss of interaction between M and dynamin. The infectious cycle of the mutant virus M.L4A was blocked at a late stage, resulting in a quasi-absence of bullet-shaped viruses in the process of budding at the cell membrane. This was associated with an accumulation of nucleocapsids at the periphery of the cell and a different pattern of VSV glycoprotein localization. Finally, we showed that M-dynamin interaction affects clathrin-dependent endocytosis. Our study suggests that hijacking the endocytic pathway might be an important feature for enveloped virus assembly and budding at the plasma membrane.

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

1998 ◽  
Vol 72 (10) ◽  
pp. 8413-8419 ◽  
Author(s):  
Maryam Ahmed ◽  
Douglas S. Lyles
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Gene Mutations ◽  
Rna Polymerases ◽  
5S Rrna ◽  
M Protein ◽  
Viral Gene ◽  
M Gene ◽  
Vesicular Stomatitis

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.

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

2007 ◽  
Vol 88 (9) ◽  
pp. 2559-2567 ◽  
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.

scholarly journals Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

2008 ◽  
Vol 82 (11) ◽  
pp. 5536-5547 ◽  
Author(s):  
B. Dancho Swinteck ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Virus Assembly ◽  
M Protein ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
N Protein ◽  
Vesicular Stomatitis ◽  
Plasma Membrane Microdomains

ABSTRACT Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

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