Selection of 3′-Template Bases and Initiating Nucleotides by Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase

Abstract

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the ؉1, ؉2, or ؉3 position numbered from the 3 end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the ؉2 and ؉3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the ؉1 position and to maintain the integrity of the 3 end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus. It encodes a single large polyprotein that is subsequently processed into at least 10 separate viral proteins by host as well as virally encoded proteases (17, 21). Of these proteins, nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is responsible for replication of viral RNA genome. Because NS5B is unique to the virus, it is a potential target for developing antiviral drugs and is being subjected to intense research. Recently, several crystal structures of NS5B have been reported, revealing several unique structural features, including a fully encircled active site and a ␤-hairpin structure in the thumb subdomain protruding towards the active site (1, 2, 9). Efforts have been made to characterize NS5B RdRp functions and to understand the mechanistic differences between NS5B and cellular polymerases. This information would help to design direct and specific inhibitors for HCV replication.HCV NS5B is believed to initiate RNA synthesis by a de novo mechanism. De novo initiation of RNA synthesis is common for most v...

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

“…This abundance of di-or trinucleotides in the gel-based assays reported herein as well as by another group (28) suggest that the RdRp may dissociate rapidly from the RNA template rather than transition efficiently into elongative synthesis. This process of abortive initiation is not uncommon and has been described recently for T7 RNA polymerase (29 -34), such that initiation complexes release short RNA templates prior to transition toward forming a stable elongative complex.…”

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

“…Reactions with concentrations of PP i that exceeded 500 ⌴ showed decreases in primer rescue, presumably by depleting concentrations of free divalent metal ions required for catalysis. Small RNA products were also resistant to PP i -mediated excision, which probably reflects the frequent dissociation of the NS5B-RNA complex during early stages of RNA synthesis (10,15,35). Stability of the elongation complex.…”

Structure–function analysis of the 3′ stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

“…However, there are conflicting descriptions of the precise site of initiation of negative-strand transcription on virusspecific templates (Oh et al 2000;Hong et al 2001;Kim et al 2002;Shim et al 2002). Although the 3ЈNTR is both highly conserved and rich in structure (Yamada et al 1996;Blight and Rice 1997;Ito and Lai 1997), purified, recombinant NS5B protein binds to the upstream NS5B coding sequence with significantly greater affinity than it does to this segment of the viral genomic RNA (Kim et al 2002).…”