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 A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

2005 ◽  
Vol 79 (12) ◽  
pp. 7327-7337 ◽  
Author(s):  
Valery Z. Grdzelishvili ◽  
Sherin Smallwood ◽  
Dallas Tower ◽  
Richard L. Hall ◽  
D. Margaret Hunt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vesicular Stomatitis Virus ◽  
In Vitro Transcription ◽  
Single Amino Acid ◽  
Wild Type ◽  
L Protein ◽  
Critical Residue ◽  
Single Amino Acid Change ◽  
Vesicular Stomatitis

ABSTRACT The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-N7 and 2′-O-adenosine positions (7mGpppAm). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5′-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.

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 Formation of Guanosine(5′)Tetraphospho(5′)Adenosine Cap Structure by an Unconventional mRNA Capping Enzyme of Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (15) ◽  
pp. 7729-7734 ◽  
Author(s):  
Tomoaki Ogino ◽  
Amiya K. Banerjee
Keyword(s):  
Vesicular Stomatitis Virus ◽  
In Vitro Transcription ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
L Protein ◽  
Transcription System ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis

ABSTRACT The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5′-cap core structure, guanosine(5′)triphospho(5′)adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5′)tetraphospho(5′)adenosine (GppppA), that is formed by the transfer of the 5′-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the γ-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

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 5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Minako Ogino ◽  
Tomoaki Ogino
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Antiviral Agents ◽  
Host Cells ◽  
Hydroxyl Group ◽  
L Protein ◽  
Acceptor Specificity ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme.

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 S-Adenosyl Homocysteine-Induced Hyperpolyadenylation of Vesicular Stomatitis Virus mRNA Requires the Methyltransferase Activity of L Protein

2008 ◽  
Vol 82 (24) ◽  
pp. 12280-12290 ◽  
Author(s):  
Summer E. Galloway ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Competitive Inhibitor ◽  
Recombinant Viruses ◽  
L Protein ◽  
Mrna Capping ◽  
Adenosyl Methionine ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
L Proteins

ABSTRACT There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3′ end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5′ cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM. To investigate this unusual phenotype, we assayed the effects of SAH on transcription using a panel of recombinant viruses that contained mutations in domain VI of the VSV L protein. The L proteins we investigated displayed a range of 5′ cap methyltransferase activities. In the present study, we show that the ability of the VSV L protein to catalyze methyl transfer correlates with its sensitivity to SAH with respect to polyadenylation, thereby indicating an intriguing connection between 5′ and 3′ end mRNA modifications. We also identified an L protein mutant that hyperpolyadenylates mRNA irrespective of the presence or absence of exogenous SAH. Further, the data presented here show that the wild-type L protein hyperpolyadenylates a percentage of VSV mRNAs in infected cells as well as in vitro.

Sign in / Sign up

Export Citation Format

Share Document