Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3 cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3 single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.Hepatitis C virus (HCV), a plus-strand RNA virus, is estimated to infect up to 3% of the world's population (44), causing liver cirrhosis and hepatocellular carcinoma (14). Following entry into the infected cell, the viral RNA directs the translation of a polyprotein that is proteolytically processed to produce 10 individual structural and nonstructural proteins (15, 32). Nonstructural protein 5B (NS5B) is at the C terminus of the polyprotein. NS5B is an RNA-dependent RNA polymerase (RdRp). Based on the paradigms of other RNA virus replication strategies (8), NS5B, along with viral and cellular proteins, forms a replicase that replicates the HCV genome. At present, functional HCV replicase has not been demonstrated in vitro. Therefore, studies of HCV RNA synthesis have focused on recombinant NS5B.Recombinant HCV NS5B can catalyze a number of reactions. In the presence of a primer-template duplex, NS5B catalyzes template-dependent but relatively nonspecific RNA synthesis (5, 23-25, 45, 46). In addition, NS5B has recently been reported to direct de novo (oligonucleotide primer-independent) synthesis (26, 30, 47), a mechanism used for the replication of many plus-strand RNA viruses (8). De novo initiation of RNA synthesis may be especially relevant for HCV since, to our knowledge, it does not contain a VPg-like protein that could mediate protein-primed RNA synthesis, and there is no evidence for a cap-snatching mechanism (32). De novo RNA synthesis directed by HCV NS5B prefers a cytidylate template and the substrate nucleotide GTP (26, 42), although ATP can also be used as an initiation nucleotide (29,42,47). In general, RNA polymerases have a higher K m for the initiation nucleotide than for the same nucleotide during elongating RNA synthesis (for examples, see references ...

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

“…Like poliovirus (15), the HCV RdRp is capable of initiating viral RNA synthesis in vitro by a primer-dependent mechanism (9,10,16). However, Flaviviridae RdRps have also been shown to initiate RNA synthesis by a de novo mechanism (12,13,(17)(18)(19). De novo initiation of virus replication is the likely preferred mechanism for HCV in infected cells (20,21).…”

“…RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12)(13)(14), although in vitro it has been shown to lack specificity for viral RNA because it readily utilizes heterologous nonviral templates (9). Like poliovirus (15), the HCV RdRp is capable of initiating viral RNA synthesis in vitro by a primer-dependent mechanism (9,10,16).…”

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

“…Initial cis cleavage through NS2-NS3 releases the NS3 protein, which in turn continues to process the precursor. Promising compounds with the ability to inhibit the viral protease (NS3) and the polymerase (NS5B) have been identified.NS5B is a 65-kDa RNA-dependent RNA polymerase capable of initiating RNA synthesis de novo in the absence of a primer (16,17,25,27,45). Three classes of inhibitors of HCV NS5B have been developed, namely, nucleoside analogue inhibitors, nonnucleoside analogue inhibitors, and pyrophosphate (PP i ) analogues.…”

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

“…Inhibition of HCV NS5B in Primer-independent (de Novo) RdRp Assay-It was previously demonstrated that HCV NS5B can initiate the RNA synthesis utilizing the de novo mechanism (41)(42)(43). Using the full-length ϳ9500-nucleotide HCV subgenomic replicon RNA template, we examined the effect of mAbs in de novo RdRp assays.…”

“…Because the copy-back or self-priming RNA synthesis by itself is incapable of generating the precise viral genome 5Ј and 3Ј termini, de novo initiation of viral replication is the most likely mechanism for HCV in infected cells (41,48,49). Moreover, in cells harboring HCV subgenomic replicons (e.g.…”