Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

Fullerton, S.W.B.; Blaschke, Martina; Coutard, Bruno; Gebhardt, Julia; Gorbalenya, Alexander E.; Canard, Bruno; Tucker, Paul A.; Rohayem, Jacques

doi:10.1128/jvi.01462-06

Cited by 65 publications

(71 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The rotation of this helix appears to be coupled to the movement of the C-terminal tail away from the active site cleft. In the unbound conformation of NV and Sapovirus RdRPs, the proximal portion of the C-terminal tail (residues 489 -502 in NV) packs against this central helix, acting as a brace to resist the outward rotation of the helix seen in the RNA-bound conformation (11,19). The movement of the C-terminal tail away from the active site cleft allows the central helix the room to rotate and interact with the primer strand and minor groove of the primer-template duplex.…”

Section: Resultsmentioning

confidence: 99%

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

Zamyatkin

Parra

Alonso

et al. 2008

Journal of Biological Chemistry

140

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

Section: Resultsmentioning

confidence: 99%

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

Zamyatkin

Parra

Alonso

et al. 2008

Journal of Biological Chemistry

140

187

View full text Add to dashboard Cite

show abstract

“…Among those, the RdRp is predicted to play an essential role being involved in the synthesis of antiviral genomic RNA, but also subgenomic RNA. Recently, the crystal structure of prototype strains of NV, sapovirus and lagovirus RdRp (Fullerton et al, 2007;Ng et al, 2004) were determined. The NV NS7 RdRp resembles the right-hand configuration of many DNA and RNA polymerases (Biswal et al, 2005;Choi et al, 2004;Lesburg et al, 1999;Ng et al, 2002Ng et al, , 2004Thompson & Peersen, 2004).…”

Section: Discussionmentioning

confidence: 99%

The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity

Högbom

Jäger²,

Robel³

et al. 2009

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

Norovirus (NV) is a leading cause of gastroenteritis worldwide and a major public health concern. So far, the replication strategy of NV remains poorly understood, mainly because of the lack of a cell system to cultivate the virus. In this study, the function and the structure of a key viral enzyme of replication, the RNA-dependent RNA polymerase (RdRp, NS7), was examined. The overall structure of the NV NS7 RdRp was determined by X-ray crystallography to a 2.3 Å (0.23 nm) resolution (PDB ID 2B43), displaying a right-hand fold typical of the template-dependent polynucleotide polymerases. Biochemical analysis evidenced that NV NS7 RdRp is active as a homodimer, with an apparent K d of 0.649 mM and a positive cooperativity (Hill coefficient n H 51.86). Crystals of the NV NS7 homodimer displayed lattices containing dimeric arrangements with high shape complementarity statistics. This experimental data on the structure and function of the NV RdRp may set the cornerstone for the development of polymerase inhibitors to control the infection with NV, a medically relevant pathogen.

show abstract

“…Indeed, terminal transferase activity is apparently widespread, having been reported for other positive-strand RNA viruses, a double-stranded RNA virus and a negative-strand RNA virus (Fullerton et al, 2007;Poranen et al, 2008;Rohayem et al, 2006;Smallwood & Moyer, 1993), suggesting that it might be a common mechanism to facilitate terminal repair.…”

Section: Terminal Transferase and Poly(a) Tail Repair Mechanismsmentioning

confidence: 94%

How RNA viruses maintain their genome integrity

Barr

Fearns

2010

Journal of General Virology

View full text Add to dashboard Cite

Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

Cited by 65 publications

References 61 publications

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

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

The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity

How RNA viruses maintain their genome integrity

Contact Info

Product

Resources

About