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 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 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 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.

scholarly journals Insertion of Enhanced Green Fluorescent Protein in a Hinge Region of Vesicular Stomatitis Virus L Polymerase Protein Creates a Temperature-Sensitive Virus That Displays No Virion-Associated Polymerase Activity In Vitro

2009 ◽  
Vol 83 (23) ◽  
pp. 12241-12252 ◽  
Author(s):  
John B. Ruedas ◽  
Jacques Perrault
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Polymerase Activity ◽  
Temperature Sensitive ◽  
Viral Mrna ◽  
L Protein ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

ABSTRACT The RNA-dependent RNA polymerase of viruses belonging to the order Mononegavirales is part of a large multifunctional L protein that also catalyzes viral mRNA capping and cap methylation. The L protein of this diverse group of agents displays six blocks of conserved sequences. The precise relationship between these conserved regions and individual functions is largely unknown, except for “domain” VI that clearly encodes a viral mRNA cap methylase. The L protein of morbilliviruses (family Paramyxoviridae) was reported to tolerate insertion of the enhanced green fluorescent protein (EGFP) in a region just upstream of domain VI. Recombinant viruses with this insertion grow well in cell culture but are highly attenuated in animal hosts. We show here that the L protein of vesicular stomatitis virus (VSV), the prototype of the Rhabdoviridae family, also tolerates insertion of EGFP at a similar site. The modified protein (LEGFP) and the resultant recombinant virus both demonstrated a sharp temperature-sensitive phenotype for polymerase activity, with reduced activity at 37°C and no activity at 37.5°C. Neither translation nor methylation of mutant virus transcripts was affected at 37°C. Curiously, mutant virus grown at permissive temperature contained about threefold-less L protein than the wild-type virus did and displayed no virion-associated polymerase activity in vitro. These findings support the notion that a flexible “hinge” region separates the cap methylase domain of L proteins from upstream functions and open up a number of avenues for studies of L-protein function in the more-tractable VSV model system.

scholarly journals Vesicular Stomatitis Virus mRNA Capping Machinery Requires Specific cis-Acting Signals in the RNA

2007 ◽  
Vol 81 (20) ◽  
pp. 11499-11506 ◽  
Author(s):  
Jennifer T. Wang ◽  
Lauren E. McElvain ◽  
Sean P. J. Whelan
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Translation Strategies ◽  
Cis Acting ◽  
Specific Manner ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis ◽  
Rna Capping ◽  
Negative Sense

ABSTRACT Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5′ end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.

scholarly journals Preferential Translation of Vesicular Stomatitis Virus mRNAs Is Conferred by Transcription from the Viral Genome

2006 ◽  
Vol 80 (23) ◽  
pp. 11733-11742 ◽  
Author(s):  
Zackary W. Whitlow ◽  
John H. Connor ◽  
Douglas S. Lyles
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Host Protein ◽  
Translation Efficiency ◽  
Viral Mrnas ◽  
Translation Inhibition ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

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.

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