Control of Template Positioning during de Novo Initiation of RNA Synthesis by the Bovine Viral Diarrhea Virus NS5B Polymerase

Abstract

The RNA-dependent RNA polymerase of the hepatitis C virus and thebovineviraldiarrheavirus(BVDV)isabletoinitiateRNAsynthesis de novo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3-end of the template can remain opposite to the 5-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotideprimed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.Despite structural and functional differences among various viral polymerases, previous biochemical and crystallographic data suggested that several distinct steps define a minimum, general mechanism of DNA or RNA synthesis (1-4). These steps involve binding of the nucleoside triphosphate, a conformational change that traps the nucleotide substrate, formation of the first phosphodiester bond, and the release of pyrophosphate. The addition of the next nucleotide requires that the polymerase translocates a single position further downstream relative to the primer/template. This movement clears the nucleotide-binding site in a processive mode of polymerization before the enzyme dissociates from its nucleic acid substrate. Such a mechanism is likely to be relevant during elongation of a growing DNA or RNA chain; however, the initiation of the reaction is often more complex. Initiation in the absence of a primer, referred to as de novo initiation, is of particular interest in this regard.Viral RNA-dependent RNA polymerases (RdRps) 4 that belong to members of the Flaviviridae family, which includes the hepatitis C virus (HCV) and the related bovine viral diarrhea virus (BVDV), were shown to initiate RNA synthesis de novo (5-12). Both HCV and BVDV contain a plus-stranded RNA genome, whi...

Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis

“…that the initiation platform might be provided by this GTP rather than directly by the Pestivirus polymerase. This hypothesis has been confirmed to some extent by D'Abramo et al (44), who found that this GTP seemingly stabilizes the interaction between the 3Ј end of the template and the priming nucleotide. A similar phenomenon could also explain our observations with the HCV-NS5b S556K mutants.…”

Hepatitis C Virus RNA Replication

“…It, furthermore, requires a "platform" to support the first nucleotide, which has to move out of the active center upon addition of the third base. This platform has not been clearly identified in the structure of the polymerase, but it might be provided by the C-terminal linker sequence (Harrus et al 2010) or by the beta flap in coordination with a GTP bound close to the active site, as suggested for pestiviruses (Lescar and Canard 2009;Choi et al 2004;D'Abramo et al 2006). Such a role of coordinating GTP in stabilizing the initiation complex might also explain the strong stimulating effect of high GTP concentration on de novo initiation of NS5B in vitro (Lohmann et al 1999b;Harrus et al 2010;Ranjith-Kumar et al 2003).…”

Bluetongue Virus VP1 Polymerase Activity In Vitro: Template Dependency, Dinucleotide Priming and Cap Dependency

“…Polymerase activities of some viruses, such as Influenza virus, Bovine viral diarrhea virus, Hepatitis C virus and GB virus-B, are initiated by dinucleotides due to priming the transcription initiation [41], [53], [54], [55]. Additionally, the study using rotavirus open cores showed that GpG, which complements the sequences of the 3′ end of rotavirus G8 ‘plus’ strand RNA, forms initiation complexes with VP1 and template ssRNA to initiate ‘minus’ strand synthesis [32].…”