Nucleotide sequence analysis of the L gene of vesicular stomatitis virus (New Jersey serotype): Identification of conserved domains in L proteins of nonsegmented negative-strand RNA viruses

ABSTRACTViruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of theRhabdoviridae,Paramyxoviridae, andFiloviridaeshare sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses.IMPORTANCENegative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses.

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Nucleotide Sequence Analysis of the Simian Virus 41 Gene Encoding the Large (L) Protein and Construction of a Phylogenetic Tree for the L Proteins of Paramyxoviruses

Journal of General Virology ◽

10.1099/0022-1317-73-10-2743 ◽

1992 ◽

Vol 73 (10) ◽

pp. 2743-2750 ◽

Cited By ~ 8

Author(s):

M. Ogawa ◽

N. Mutsuga ◽

M. Tsurudome ◽

M. Kawano ◽

H. Matsumura ◽

...

Keyword(s):

Sequence Analysis ◽

Nucleotide Sequence ◽

Phylogenetic Tree ◽

Simian Virus ◽

Nucleotide Sequence Analysis ◽

Gene Encoding ◽

L Protein ◽

L Proteins

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A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

Journal of Virology ◽

10.1128/jvi.00146-18 ◽

2018 ◽

Vol 92 (8) ◽

pp. e00146-18 ◽

Cited By ~ 5

Author(s):

Ryan H. Gumpper ◽

Weike Li ◽

Carlos H. Castañeda ◽

M. José Scuderi ◽

James K. Bashkin ◽

...

Keyword(s):

Crystal Structure ◽

Vesicular Stomatitis Virus ◽

Ebola Virus ◽

Rna Viruses ◽

Rna Virus ◽

Viral Rna ◽

Negative Strand ◽

Double Stranded Dna ◽

Vesicular Stomatitis ◽

Syncytial Virus

ABSTRACTPolyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

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Adding Genes to the RNA Genome of Vesicular Stomatitis Virus: Positional Effects on Stability of Expression

Journal of Virology ◽

10.1128/jvi.76.15.7642-7650.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7642-7650 ◽

Cited By ~ 62

Author(s):

Gail W. Wertz ◽

Robin Moudy ◽

L. Andrew Ball

Keyword(s):

Gene Expression ◽

Vesicular Stomatitis Virus ◽

Rna Viruses ◽

Transcriptional Unit ◽

N Gene ◽

Wild Type ◽

Control Of Gene Expression ◽

Negative Strand ◽

Transcriptional Termination ◽

Vesicular Stomatitis

ABSTRACT Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.

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