scholarly journals Vesicular Stomatitis Viruses Resistant to the Methylase Inhibitor Sinefungin Upregulate RNA Synthesis and Reveal Mutations That Affect mRNA Cap Methylation

2007 ◽  
Vol 81 (8) ◽  
pp. 4104-4115 ◽  
Author(s):  
Jianrong Li ◽  
John S. Chorba ◽  
Sean P. J. Whelan
Keyword(s):  
Gene Expression ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Gene ◽  
Negative Strand ◽  
L Protein ◽  
Conserved Sequence ◽  
Viral Yield ◽  
Vesicular Stomatitis

ABSTRACT Sinefungin (SIN), a natural S-adenosyl-l-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2′-O (2′-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2′-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.

scholarly journals Structural Properties of the C Terminus of Vesicular Stomatitis Virus N Protein Dictate N-RNA Complex Assembly, Encapsidation, and RNA Synthesis

2012 ◽  
Vol 86 (16) ◽  
pp. 8720-8729 ◽  
Author(s):  
Bianca S. Heinrich ◽  
Benjamin Morin ◽  
Amal A. Rahmeh ◽  
Sean P. J. Whelan
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
C Terminus ◽  
Vesicular Stomatitis ◽  
Rna Complex

The vesicular stomatitis virus (VSV) nucleoprotein (N) associates tightly with the viral genomic RNA. This N-RNA complex constitutes the template for the RNA-dependent RNA polymerase L, which engages the nucleocapsid via its phosphoprotein cofactor P. While N and P proteins play important roles in regulating viral gene expression, the molecular basis of this regulation remains incompletely understood. Here we show that mutations in the extreme C terminus of N cause defects in viral gene expression. To determine the underlying cause of such defects, we examined the effects of the mutations separately on encapsidation and RNA synthesis. Expression of N together with P inEscherichia coliresults predominantly in the formation of decameric N-RNA rings. In contrast, nucleocapsid complexes containing the substitution NY415Aor NK417Awere more loosely coiled, as revealed by electron microscopy (EM). In addition, the NEF419/420AAmutant was unable to encapsidate RNA. To further characterize these mutants, we engineered an infectious cDNA clone of VSV and employed N-RNA templates from those viruses to reconstitute RNA synthesisin vitro. The transcription assays revealed specific defects in polymerase utilization of the template that result in overall decreased RNA quantities, including reduced amounts of leader RNA. Passage of the recombinant viruses in cell culture led to the accumulation of compensatory second-site mutations in close proximity to the original mutations, underscoring the critical role of structural features within the C terminus in regulating N function.

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

2002 ◽  
Vol 76 (15) ◽  
pp. 7642-7650 ◽  
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.

scholarly journals Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein

2009 ◽  
Vol 83 (21) ◽  
pp. 11043-11050 ◽  
Author(s):  
Amal A. Rahmeh ◽  
Jianrong Li ◽  
Philip J. Kranzusch ◽  
Sean P. J. Whelan
Keyword(s):  
Amino Acid ◽  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Amino Acid Substitutions ◽  
Individual Amino Acid ◽  
L Protein ◽  
C Terminus ◽  
Methylation Assay ◽  
Vesicular Stomatitis

ABSTRACT During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2′-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2′-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2′-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2′-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2′-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2′-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2′-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5′ terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2′-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2′-O and G-N-7 methylation of the cap structure.

scholarly journals Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis

2008 ◽  
Vol 83 (4) ◽  
pp. 1930-1940 ◽  
Author(s):  
Jianrong Li ◽  
Amal Rahmeh ◽  
Vesna Brusic ◽  
Sean P. J. Whelan
Keyword(s):  
Amino Acid ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Full Length ◽  
Single Amino Acid ◽  
Mrna Synthesis ◽  
L Protein ◽  
Vesicular Stomatitis ◽  
Opposing Effects ◽  
Gene Junction

ABSTRACT The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3′-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3′ termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5′ cap formation and 3′ polyadenylation.

scholarly journals HCMV exploits STING signaling and counteracts IFN and ISG induction to facilitate dendritic cell infection

2022 ◽  
Author(s):  
Bibiana Costa ◽  
Jennifer Becker ◽  
Tobias Krammer ◽  
Felix Mulenge ◽  
Verónica Durán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Gene Expression ◽  
Productive Infection ◽  
Viral Gene ◽  
Human Pathogen ◽  
Cell Infection ◽  
Complex Interactions ◽  
Life Threatening ◽  
Immunocompromised Hosts

Abstract Human cytomegalovirus (HCMV) is a widespread obligatory human pathogen causing life-threatening disease in immunocompromised hosts. Myeloid cells such as monocyte-derived dendritic cells (moDCs) are targets of HCMV. Here, we performed single-cell RNA sequencing, which revealed infection of most moDCs upon in vitro HCMV exposure, whereas only a fraction of them initiated viral gene expression. We identified three moDC subsets, of which CD1a−/CD86− cells showed the highest susceptibility. Upon HCMV entry, STING activation not only induced IFN-β, but also promoted viral gene expression. Upon progression of infection, IFN-β but not IFN-λ1 expression was inhibited. Similarly, ISG expression was initially induced and then shut off and thus allowed productive infection. Increased viral gene expression was associated with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Thus, moDC permissiveness to HCMV depends on complex interactions between virus sensing, regulation of IFNs/ISGs and viral gene expression.

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.

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