scholarly journals Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA

2004 ◽  
Vol 101 (16) ◽  
pp. 5952-5957 ◽  
Author(s):  
K. R. Qanungo ◽  
D. Shaji ◽  
M. Mathur ◽  
A. K. Banerjee
Keyword(s):  
Rna Polymerase ◽  
Vesicular Stomatitis Virus ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Transcription And Replication

scholarly journals Regulation of RNA Synthesis by the Genomic Termini of Vesicular Stomatitis Virus: Identification of Distinct Sequences Essential for Transcription but Not Replication

1999 ◽  
Vol 73 (1) ◽  
pp. 297-306 ◽  
Author(s):  
Sean P. J. Whelan ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Virus ◽  
N Gene ◽  
Specific Sequence ◽  
Virus Identification ◽  
Sequence Elements ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Sequence Requirements ◽  
Transcription And Replication

ABSTRACT The RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV), a nonsegmented negative-strand RNA virus, directs two discrete RNA synthetic processes, transcription and replication. Available evidence suggests that the two short extragenic regions at the genomic termini, the 3′ leader (Le) and the complement of the 5′ trailer (TrC), contain essential signals for these processes. We examined the roles in transcription and replication of sequences in Le and TrC by monitoring the effects of alterations to the termini of subgenomic replicons, or infectious viruses, on these RNA synthetic processes. Distinct elements in Le were found to be required for transcription that were not required for replication. The promoter for mRNA transcription was shown to include specific sequence elements within Le at positions 19 to 29 and 34 to 46, a separate element at nucleotides 47 to 50, the nontranscribed leader-N gene junction. The sequence requirements for transcription within the Le region could not be supplied by sequences found at the equivalent positions in TrC. In contrast, sequences from either Le or TrC functioned well to signal replication, indicating that within the confines of the VSV termini, the sequence requirements for replication were less stringent. Deletions engineered at the termini showed that the terminal 15 nucleotides of either Le or TrC allowed a minimal level of replication. Within these confines, levels of replication were affected by both the extent of complementarity between the genomic termini and the involvement of the template in transcription. In agreement with our previous observations, increasing the extent of complementarity between the natural termini increased levels of replication, and this effect was most operative at the extreme genome ends. In addition, abolishing the use of Le as a promoter for transcription enhanced replication. These analyses (i) identified signals at the termini required for transcription and replication and (ii) showed that Le functions as a less efficient promoter for replication than TrC at least in part because of its essential role in transcription. Consequently, these observations help explain the asymmetry of VSV replication which results in the synthesis of more negative- than positive-sense replication products in infected cells.

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.

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus

1982 ◽  
Vol 2 (1) ◽  
pp. 66-75
Author(s):  
S Gillies ◽  
V Stollar
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Aedes Albopictus ◽  
Rna Synthesis ◽  
Cytopathic Effect ◽  
Viral Gene ◽  
Inhibition Of Protein Synthesis ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Aedes Albopictus Cells

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

scholarly journals Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

2015 ◽  
Vol 89 (21) ◽  
pp. 11019-11029 ◽  
Author(s):  
Frauke Beilstein ◽  
Linda Obiang ◽  
Hélène Raux ◽  
Yves Gaudin
Keyword(s):  
T Cells ◽  
Immune System ◽  
Mhc Class I ◽  
Vesicular Stomatitis Virus ◽  
Cd8 T Cells ◽  
Matrix Protein ◽  
Catalytic Subunit ◽  
The Matrix ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACTThe matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system.IMPORTANCEThe immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

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