Two Crucial Early Steps in RNA Synthesis by the Hepatitis C Virus Polymerase Involve a Dual Role of Residue 405

Abstract

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5B initiates RNA synthesis by a de novo mechanism and then processively copies the whole RNA template. Dissections of de novo RNA synthesis by genotype 1 NS5B proteins previously established that there are two successive crucial steps in de novo initiation. The first is dinucleotide formation, which requires a closed conformation, and the second is the transition to elongation, which requires an opening of NS5B. We also recently published a combined structural and functional analysis of genotype 2 HCV-NS5B proteins (of strains JFH1 and J6) that established residue 405 as a key element in de novo RNA synthesis ( T he hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (28). The genome of HCV is a 9,600-nucleotide (nt)-long RNA molecule encompassing a single open reading frame (ORF) that is translated primarily into one polyprotein and flanked by nontranslated regions (NTRs). The NTRs are the most conserved parts of the viral genome and play important roles in viral translation and replication. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different mature proteins. The nonstructural proteins NS3 to NS5B are associated within a membrane replication complex in which NS5B is the RNA-dependent RNA polymerase (RdRp). NS5B copies the RNA genome into a complementary negative strand and subsequently uses this negative strand as the template for the synthesis of new HCV genomes (for a review, see reference 21).In vitro, recombinant NS5B is a highly processive RdRp that copies RNA templates up to the size of the HCV genome (4). C-terminal deletions of the 21-residue transmembrane helix (NS5B⌬C21) are fully active and much easier to produce and purify than the full-length enzyme (37), so that these deletions are used almost exclusively for structural and functional characterization of NS5B. These studies have shown that NS5B can initiate RNA synthesis by a de novo mechanism (19) allowing full copy of the template in a single polymerization round (14,29). De novo initiation from the 3= end of the viral positive-and negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells (8). Recombinant NS5B is very poorly active overall despite its high processivity and a turnover number comparable to that of other polymerases (7). Dissections of the early steps of RNA synthesis in vitro (12,14,29) have shown that this is due to two inefficient steps in de novo RNA synthesis by NS5B; the first step is de novo initiation proper, i.e., formation of the first dinucleotide complementary to the last two 3= bases of the template, and the second step is use of this dinucleotide as a primer for further RNA synthesis.Structural analysis of NS5B led us to propose that these two steps are closely linked to two conformational st...

Molecular and Structural Basis for the Roles of Hepatitis C Virus Polymerase NS5B Amino Acids 15, 223, and 321 in Viral Replication and Drug Resistance

“…The two rate-limiting steps during de novo RNA synthesis have been identified as the synthesis of the first dinucleotide and the transition from initiation to elongation, which requires rearrangement of NS5B (6). Therefore, it is possible that the increase in our de novo RNA synthesis activity resulted from enhancement of a rate-limiting step by the triple-mutant enzyme with a conformation compatible with RNA binding (7), which is consistent with the observation that JFH-1 is able to synthesize the first dinucleotide and transition to elongation more efficiently than other isolates (20). The molecular mechanism of how this conformation could confer resistance to 2=-F-2=-C-methylguanosine triphosphates remains unclear.…”

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

“…These contacts are believed to restrict the flexibility of the subdomains and favor the first steps, or initiation, of RNA synthesis leading to the formation of the primer strand. Therefore, it is believed that primer extension by NS5B during the elongation step requires important structural changes involving an opening of the thumb and fingers (7,8). The capacity to experimentally trap and characterize the elongation complex (EC) has been a significant milestone to help understand replication of RNA by HCV NS5B (9, 10).…”

Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

“…Although the authors of that study suggested that this resulted in an initiationincompetent enzyme, our results indicate that filibuvir and lomibuvir in fact increase the initiation (de novo) but inhibit the elongation activity of the G1b RdRp. Recently, recombinant HCV RdRp was proposed to exist as a mixture of conformations which are in dynamic equilibrium, with stabilization of one conformation occurring at the expense of the other in solution (45). Given that formation of the first few phosphodiester bonds and the transition to elongation are two known rate-limiting steps of the HCV RdRp reaction (44,70,71), it is likely that T2 and P-␤ enhance the initiation efficiency (de novo) of the HCV RdRp by stabilizing the RdRp conformations required for these rate-limiting steps.…”

“…A "closed" conformation, facilitated by the interactions between the finger and thumb domains, allows the de novo formation of a dinucleotide for replication initiation (44). The more relaxed "open" conformation of the HCV RdRp is formed by displacement of the ␤-hairpin loop as well as a C-terminal segment just upstream of the transmembrane domain, called the linker (45,46). This open conformational change allows room for the nascent double-stranded RNA (dsRNA) and is thought to be responsible for the primer-extension activity of the RdRp (45,46).…”

“…The more relaxed "open" conformation of the HCV RdRp is formed by displacement of the ␤-hairpin loop as well as a C-terminal segment just upstream of the transmembrane domain, called the linker (45,46). This open conformational change allows room for the nascent double-stranded RNA (dsRNA) and is thought to be responsible for the primer-extension activity of the RdRp (45,46). Studies involving NNIs and recombinant RdRps have focused primarily on the ability of the NNIs to inhibit the primer-dependent activity of the HCV RdRp, and little is currently known on what effect NNIs have on de novo RdRp activity.…”