Vesicular stomatitis virus N and NS proteins form multiple complexes

Self Cite

127

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

Section: Discussionmentioning

confidence: 79%

Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs

Pattnaik

Wertz

1990

Self Cite

127

“…Our previous experience of the sensitivity of VSV RNA replication to the N:P:L protein ratios led us to anticipate that some of the gene rearrangements would be lethal (28). In particular, we expected severe phenotypic consequences to result from both the underexpression of P protein, which acts as a chaperone to incorporate N into assembling nucleocapsids (10,16,19), and the overexpression of M protein, which inhibits viral transcription (8,9,21) and condenses the viral nucleocapsids in preparation for particle formation (22,24). Although more severe perturbations of the levels of viral proteins would doubtless have more profound effects on the viral phenotype, variant MGP, which combined these features, replicated in cell culture about as well as the wild-type virus and had a similar LD 50 in mice ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Phenotypic Consequences of Rearranging the P, M, and G Genes of Vesicular Stomatitis Virus

Ball

Pringle

Flanagan

et al. 1999

111

The nonsegmented negative-strand RNA viruses (orderMononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3′ end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3′ promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.

“…The active polymerase complex of vesicular stomatitis virus (VSV) consists of the phosphoprotein (P) (formerly referred to by the misnomer nonstructural [NS] protein, more recently renamed P [23]) associated with the polymerase L protein and the nucleocapsid (N) protein RNA (N-RNA) template (3,15). Both the P and N proteins are essential for transcription, replication, and encapsidation of the viral genome (2,12,30,31,37,38,39). The P protein facilitates L protein contact with the N-RNA template during transcription and allows chain elongation (13,14,20).…”

mentioning

confidence: 99%

Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey

Bilsel

Rowe

Fitch

et al. 1990

The entire phosphoprotein (P) and nucleocapsid (N) protein gene sequences and deduced amino acid sequences for 18 selected vesicular stomatitis virus isolates representative of the natural genetic diversity within the New Jersey serotype are reported. Phylogenetic analysis of the data using maximum parsimony allowed construction of evolutionary trees for the individual genes and the combined N, P, and glycoprotein (G) genes of these viruses. Virtually identical rates of nucleotide substitutions were found for each gene, indicating that evolution of these genes occurs at essentially the same rate. Although up to 19 and 17% sequence differences were evident in the P and N genes, respectively, no variation in gene length or evidence of recombinational rearrangements was found. However, striking evolutionary differences were observed among the amino acid sequences of vesicular stomatitis virus New Jersey N, P, and G proteins. The N protein amino acid sequence was the most highly conserved among the different isolates, indicating strong functional and structural constraints. Conversely, the P protein amino acid sequences were highly variable, indicating considerably fewer constraints or greater evolutionary pressure on the P protein. Much of the remarkable amino acid variability of the P protein resided in a hypervariable domain located between amino acids 153 and 205. The variability within this region would be consistent with it playing a structural role as a spacer to maintain correct conformational presentation of the separate active domains of this multifunctional protein. In marked contrast, the adjacent domain I of the P protein (previously thought to be under little evolutionary constraint) contained a highly conserved region. The colocalization of a short, potentially functional overlapping open reading frame to this region may explain this apparent anomaly.