scholarly journals The multifunctional RNA polymerase L protein of non-segmented negative strand RNA viruses catalyzes unique mRNA capping

Uirusu ◽  
2014 ◽  
Vol 64 (2) ◽  
pp. 165-178 ◽  
Author(s):  
Tomoaki OGINO
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Negative Strand ◽  
L Protein ◽  
Mrna Capping

scholarly journals Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Codon Usage ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Genomic Rna ◽  
Negative Strand ◽  
Unique Mechanism

ABSTRACTNegative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. Byin situanalyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCENegative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

scholarly journals Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses

2015 ◽  
Vol 43 (17) ◽  
pp. 8416-8434 ◽  
Author(s):  
Kathleen C. Lehmann ◽  
Anastasia Gulyaeva ◽  
Jessika C. Zevenhoven-Dobbe ◽  
George M. C. Janssen ◽  
Mark Ruben ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Covalent Binding ◽  
Equine Arteritis Virus ◽  
Structural Protein ◽  
Proteomics Analysis ◽  
Future Studies ◽  
Conserved Domain ◽  
Biochemical Assays ◽  
Mrna Capping

Abstract RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.

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 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.

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