Recombinant viral RdRps can initiate RNA synthesis from circular templates

Abstract

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage f6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the f6 RdRp could only use circular RNAs made from the 3¢-terminal sequence of the f6 genome.

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

“…We therefore rule out that the 18 base pair DNA: RNA duplex segment contributes to NS5B binding. These observations are consistent with the reported narrow binding tunnel associated with the closed conformation of NS5B, which is unable to accommodate a double stranded segment (7,38,39).…”

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

“…The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22,(47)(48)(49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45,60).…”

“…Bovine viral diarrhea virus (BVDV) RdRp was also shown to exist in a partially open conformation (11). Ranjith-Kumar and Kao (49) demonstrated that the HCV RdRp could initiate RNA synthesis from a circular RNA template, and thus, the threading of a singlestranded RNA into the template channel is not required for de novo-initiated RNA synthesis. Altogether, these results raise the possibility that the HCV RdRp can undergo rearrangements from the closed conformation seen in the crystal structure prior to de novo initiation.…”

Impaired Replication of Hepatitis C Virus Containing Mutations in a Conserved NS5B Retinoblastoma Protein-Binding Motif

“…RdRp assay mixtures contained 1 pmol of linear template RNA or 30 pmol of circular (C) template RNA and 0.04 M of NS5B protein (2,23). The assays were carried out in 20-l reaction mixtures containing 20 mM sodium glutamate (pH 8.2), 0.5 mM DTT, 4 mM MgCl 2 , 12, 0.5% (vol/vol) Triton X-100, 200 M ATP and UTP, and 250 nM [␣-32 P]CTP (MP Biomedicals).…”