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 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 Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

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 RUBELLA VIRUS CARRIER CULTURES DERIVED FROM CONGENITALLY INFECTED INFANTS

1966 ◽  
Vol 123 (5) ◽  
pp. 795-816 ◽  
Author(s):  
William E. Rawls ◽  
Joseph L. Melnick
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Persistence ◽  
Carrier State ◽  
Congenital Rubella ◽  
Infected Cells ◽  
Eclipse Phase ◽  
Vesicular Stomatitis ◽  
Simplex Virus

Spontaneous rubella carrier cultures derived from tissues of infants with congenital rubella were studied in an attempt to elucidate a possible mechanism for viral persistence observed in these infants. Chronically infected cells were found to have a reduced growth rate and the cultures appeared to have a shortened life span. The rubella carrier state was not dependent on serum inhibitors or rubella antibodies. Virtually every cell in the carrier population was found to be producing virus. The carrier cultures could not be cured by rubella antibodies. The rubella-infected cells were resistant to superinfection with vesicular stomatitis virus and herpes simplex virus but were susceptible to infection with echovirus 11. The replication of vesicular stomatitis virus was apparently blocked at an intracellular site, for the virus readily adsorbed to the chronically infected cells and entered into an eclipse phase; however no infectious virus developed. No evidence of interferon production by these cells could be obtained. It is postulated that clones of rubella-infected cells in vivo, with properties similar to those in carrier cultures developed in vitro from tissues of in utero infected infants, might explain the observed viral persistence noted in congenital rubella.

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 A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

2007 ◽  
Vol 82 (2) ◽  
pp. 775-784 ◽  
Author(s):  
Jianrong Li ◽  
Amal Rahmeh ◽  
Marco Morelli ◽  
Sean P. J. Whelan
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Single Amino Acid ◽  
L Protein ◽  
Mrna Capping ◽  
Rna Triphosphatase ◽  
Vesicular Stomatitis ◽  
L Proteins ◽  
Covalent Intermediate ◽  
Negative Sense

ABSTRACT Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5′ monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5′ diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.

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 Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

1989 ◽  
Vol 108 (3) ◽  
pp. 811-819 ◽  
Author(s):  
K Suh ◽  
J E Bergmann ◽  
C A Gabel
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Plasmid Vector ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
High Mannose

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

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.

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