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

2006 ◽  
Vol 87 (11) ◽  
pp. 3379-3384 ◽  
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.

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

2002 ◽  
Vol 76 (16) ◽  
pp. 8011-8018 ◽  
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.

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

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
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.

Association of the nucleocapsid protein N of vesicular stomatitis virus with phospholipids vesicles containing the matrix protein M

1984 ◽  
Vol 62 (11) ◽  
pp. 1174-1180 ◽  
Author(s):  
John Capone ◽  
Hara P. Ghosh
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Nucleocapsid Protein ◽  
Matrix Protein ◽  
Lipid Vesicles ◽  
Viral Envelope ◽  
M Protein ◽  
Phospholipid Vesicles ◽  
N Protein ◽  
The Matrix ◽  
Vesicular Stomatitis

The matrix protein M and the nucleocapsid protein N were isolated from vesicular stomatitis virus and reconstituted into artificial phospholipid vesicles. While the M protein could be reconstituted into phospholipid vesicles, the N protein had no affinity for lipid vesicles. The N protein could, however, associate with phospholipid vesicles in the presence of M protein. Identical results were also obtained when an in vitro system synthesizing M and N proteins was used for reconstitution. The results suggest that M protein is involved in virus maturation by interacting with the viral envelope and the N protein of the nucleoprotein core.

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 The host cellular protein Ndufaf4 interacts with the vesicular stomatitis virus M protein and affects viral propagation

2020 ◽  
Author(s):  
Wei Pan ◽  
Hongmei Wang ◽  
Hongbin He
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Cellular Protein ◽  
M Protein ◽  
Type I ◽  
Globular Domain ◽  
Viral Propagation ◽  
The Matrix ◽  
Assembly Factor ◽  
Vesicular Stomatitis

Abstract Background: Vesicular stomatitis virus (VSV) is an archetypal member of Mononegavirales which causes important diseases in cattle, horses and pigs. The matrix protein (M) of VSV plays critical roles in the replication, assembly/budding and pathogenesis of VSV. To further investigate the role of M during viral growth, we used a two-hybrid system to screen for host factors that interact with the M protein. Results: Here, NADH: ubiquinone oxidoreductase complex assembly factor 4 (Ndufaf4) was identified as an M-binding partner, and this interaction was confirmed by yeast cotransformation and GST pulldown assays. The globular domain of M was mapped and shown to be critical for the M-Ndufaf4 interaction. Two double mutations (E156A/H157A, D180A/E181A) in M impaired the M-Ndufaf4 interaction. Overexpression of Ndufaf4 inhibited VSV propagation, and knockdown of Ndufaf4 by short hairpin RNA (shRNA) markedly promoted VSV replication. Finally, we also demonstrate that the anti-VSV effect of Ndufaf4 is independent of activation of the type I IFN response. These results indicated that Ndufaf4 might exploit other mechanisms to affect VSV replication. Conclusions: In summary, we identify Ndufaf4 as a potential target for the inhibition of VSV propagation. These results provided further insight into the study of VSVpathogenesis.

scholarly journals Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system

2005 ◽  
Vol 86 (12) ◽  
pp. 3357-3363 ◽  
Author(s):  
Jérôme Solon ◽  
Olivier Gareil ◽  
Patricia Bassereau ◽  
Yves Gaudin
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Experimental System ◽  
M Protein ◽  
Extracellular Medium ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Membrane Deformations

The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.

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 The Matrix Protein of Vesicular Stomatitis Virus Inhibits Nucleocytoplasmic Transport When It Is in the Nucleus and Associated with Nuclear Pore Complexes

2000 ◽  
Vol 20 (22) ◽  
pp. 8590-8601 ◽  
Author(s):  
Jeannine M. Petersen ◽  
Lu-Shuin Her ◽  
Virgil Varvel ◽  
Elsebet Lund ◽  
James E. Dahlberg
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Nucleocytoplasmic Transport ◽  
M Protein ◽  
Single Amino Acid ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Pore Complexes

ABSTRACT The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of bidirectional nuclear transport. Here we demonstrate that inhibition occurs when M protein is in the nucleus ofXenopus laevis oocytes and that M activity is readily reversed by a monoclonal antibody (αM). We identify a region of M protein, amino acids 51 to 59, that is required both for inhibition of transport and for efficient recognition by αM. When expressed in transfected HeLa cells, M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim. Moreover, mutation of a single amino acid, methionine 51, eliminates both transport inhibition and targeting to NPCs. We propose that M protein inhibits bidirectional transport by interacting with a component of the NPC or an NPC-associated factor that participates in nucleocytoplasmic transport.

scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

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