A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases

Bruenn, Jeremy A.

doi:10.1093/nar/gkg277

Cited by 268 publications

(240 citation statements)

References 104 publications

Supporting

Mentioning

231

Contrasting

Unclassified

Order By: Relevance

“…8) (35). It has been previously proposed that the F1 motif of viral RdRps could be involved in interacting with the RNA template and/or incoming nucleotides (8,11,15). This motif has also been defined in different flavivirus RdRps (22,29).…”

Section: Resultsmentioning

confidence: 99%

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Iglesias

Filomatori

Gamarnik

2011

J Virol

View full text Add to dashboard Cite

The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5 untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.Dengue virus (DENV) is the single most significant arthropod-borne virus pathogen in humans. It belongs to the Flaviviridae family together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The World Health Organization continues reporting dengue outbreaks every year in the Americas and Asia. In spite of the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable. Although a model for DENV RNA synthesis was previously proposed (17), molecular aspects of the mechanism by which the polymerase specifically amplifies the viral genome are still unclear for DENV and other flaviviruses. To further understand this viral process, we investigated functional properties of the viral polymerase NS5.NS5 is the largest of the flavivirus proteins (105 kDa); it contains an N-terminal methyltransferase domain (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. The MTase is responsible for methylation of the cap structure present at the 5Ј end of the viral genome. This process involves methylation in two positions, guanine N-7 and ribose 2Ј-O (12, 13, 28, 38). The RdRp domain has primer independent (de novo) RNA synth...

show abstract

Section: Resultsmentioning

confidence: 99%

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Iglesias

Filomatori

Gamarnik

2011

J Virol

View full text Add to dashboard Cite

show abstract

“…Five conserved motifs, A-E (Fig. S4), with established functions in the catalytic mechanism are located in the palm domain (19). This domain is composed of two α-helices packed against one face of a four-stranded antiparallel β-sheet containing motifs A, C, and D (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, the residues 144-207 and the T helix appear to be structurally unique compared to other RdRps. In other RdRps, the F motif is found in the N terminal of their sequences, where it connects the thumb domain with the fingers domain, and the motif is possibly involved in template unwinding (19). This motif cannot be identified in the β-subunit by sequence alignment.…”

Section: Resultsmentioning

confidence: 99%

Structure of the Qβ replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

Kidmose

Vasiliev

Chetverin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qβ is composed of the viral catalytic β-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the β-subunit and the two host proteins to 2.5-Å resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the β-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the β-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.protein biosynthesis | virus

show abstract

“…2 and 4). The threedimensional arrangement of N-terminal, fingers, palm, and thumb domains, as well as the active site residues in motifs A-F are nearly universally shared (5).…”

Section: Norwalk Virus (Nv)mentioning

confidence: 99%

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Zamyatkin

Parra

Alonso

et al. 2008

Journal of Biological Chemistry

139

187

View full text Add to dashboard Cite

Crystal structures of Norwalk virus polymerase bound to an RNA primer-template duplex and either the natural substrate CTP or the inhibitor 5-nitrocytidine triphosphate have been determined to 1.8 Å resolution. These structures reveal a closed conformation of the polymerase that differs significantly from previously determined open structures of calicivirus and picornavirus polymerases. These closed complexes are trapped immediately prior to the nucleotidyl transfer reaction, with the triphosphate group of the nucleotide bound to two manganese ions at the active site, poised for reaction to the 3-hydroxyl group of the RNA primer. The positioning of the 5-nitrocytidine triphosphate nitro group between the ␣-phosphate and the 3-hydroxyl group of the primer suggests a novel, general approach for the design of antiviral compounds mimicking natural nucleosides and nucleotides. Norwalk virus (NV)4 is the prototype species of the Norovirus genus within the Caliciviridae and is a major cause of gastroenteritis outbreaks in developed countries (1). Unfortunately, effective treatments are not currently available for many important diseases caused by NV and related RNA viruses. The virally encoded RNA-dependent RNA polymerase (RdRP) is the central enzyme required for replication (2) and is one of the key targets for the development of novel antiviral agents. Recently, 5-nitrocytidine triphosphate (NCT) was identified as a potent inhibitor of picornaviral polymerases, and the nucleoside 5-nitrocytidine was found to have low toxicity and significant antiviral activity in a cultured cell viral infection model (3). A structural and mechanistic basis for rationalizing the inhibitory activity of NCT and related inhibitors is currently lacking because of a shortage of high resolution structural information on RdRP replication complexes.Details on the structure and mechanism of viral RdRPs are clearly required to understand the replication of RNA viruses and to develop more effective antiviral agents. Previous structural studies of viral RdRPs from positive strand RNA viruses and double-strand RNA viruses indicate that the general features of RdRP architecture are highly conserved throughout a diverse range of viruses (reviewed in Refs. 2 and 4). The threedimensional arrangement of N-terminal, fingers, palm, and thumb domains, as well as the active site residues in motifs A-F are nearly universally shared (5).The structural conservation seen in RdRPs suggests that the enzymatic mechanism of nucleotidyl transfer is also highly conserved. Studies primarily on poliovirus RdRP have revealed many of the basic features underlying the nucleotidyl transfer reaction in RdRPs (6, 7). These studies and others indicate that RdRPs, like other polynucleotide polymerases, follow a fivestep reaction cycle involving (i) the binding of an NTP complementary to the base of the template to form an initial "open" complex, followed by (ii) a conformational change to the "closed" complex, (iii) nucleotidyl transfer and translocation, (iv) a second confor...

show abstract

A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases

Cited by 268 publications

References 104 publications

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Structure of the Qβ replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Contact Info

Product

Resources

About