NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

Abstract

Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3′-terminal CMP to generate the dinucleoside tetraphosphate products Ap 4 C, Up 4 C, and Cp 4 C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3′deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.drug resistance | NS5B | nucleoside analog | pre-steady-state kinetics | pyrophosphorolysis C hronic infection with hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Current treatment of the infection is based on the combination of pegylated IFN-α and ribavirin, which can lead to a sustained viral response (functional cure) in ∼50% of patients infected with HCV genotype 1 and in 80% of patients infected with HCV genotype 2 or 3. With the approval of the new HCV protease inhibitors, boceprevir and telaprevir, added to pegylated IFN and ribavirin, cure rates in treatment-naive patients infected with HCV genotype 1 have improved to ∼70%. However, these treatments are associated with significant side effects, and a large number of patients either do not respond or develop resistance to the current treatment options (1-3). New drugs in development include nucleoside analogs targeting the HCV polymerase (NS5B). These compounds, in contrast to protease inhibitors, are characterized by potency across all HCV genotypes and a higher barrier to resistance (4, 5).NS5B is a 68-kDa RNA-dependent RNA polymerase (RdRp) responsible for replication of the HCV genome. Its structure has the canonic right-hand polymerase structure with thumb, fingers, and palm domains, although extensions from the fingers domain enclose the active site and a β-loop originating from the thumb prevents the binding of duplex RNA (6, 7). HCV RNA replication initiates de novo without a primer in vivo, and RNA synthesis by NS5B proceeds through two very distinct mechanistic phases: initiation and elongation. Initiation of RNA synthesis is inefficient and associated with the production of a large amount of short, abortive RNA products, which are released into solution (8-10). Following initiation, RNA elongation is rapid and processive during the elongation phase (11). The different mechanistic and kinetic properties associated with the initiation and elongation phases suggest that different conformations of the NS5B protein may be required in th...

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

“…It is generally assumed that, in the absence of any proofreading mechanism, nucleotide incorporation by RNA polymerases is irreversible. How- ever, it has recently been reported that HCV NS5B was able to excise the 3=-terminal nucleotide from the RNA (25). A suggested extension of this finding could be to measure the effect of NTPmediated excision on the stability of incorporation and the inhibition potency of chain terminators, such as those we just characterized.…”

“…The primer extension and pause reaction mixture containing 6.5 M NS5B, 20 M pGG, 20 M RNA template, 25 M ATP, and 12.5 M GTP in the optimized reaction buffer (40 mM Tris-Cl, pH 7.0, 40 mM NaCl, 5 mM DTT, and 2 mM MgCl 2 ) was run for 2 h at 30°C to generate a 9-mer, followed by the addition of 20 M CTP containing 13 nM 33 P-radiolabeled CTP, and incubated for 30 s at 30°C to generate a 10-mer. As previously discovered (9,25), all of the active EC was pelleted during the reaction and isolated by centrifugation at 16,000 ϫ g for 5 min at room temperature using a benchtop centrifuge (model 5415 D; Eppendorf). After centrifugation, the supernatant was removed and the remaining pellet was washed twice by additional resuspension in the wash buffer (40 mM Tris-Cl, pH 7.0, 20 mM NaCl, 5 mM DTT, and 2 mM MgCl 2 ) and centrifugation to remove residual contaminants.…”

Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation

“…A major advance in studying the polymerization reaction was achieved in studies showing that a 1-2-h incubation of enzyme with template, a dinucleotide (pGG), and 2 of the 4 nucleoside triphosphates (NTPs) led to the formation of a stalled, yet very stable elongation complex that catalyzed subsequent elongation reactions with processivity and rates expected for viral replication in vivo (7,9). These studies enable dissection of the kinetics of polymerization during processive elongation using transient state kinetic analysis.…”

HIV-1 reverse transcriptase and antiviral drug resistance. Part 2

“…In a fascinating parallel development, the hepatitis C (HCV) virus RNA-directed RNA polymerase (NS5B) has been shown to be capable of catalyzing NTP-mediated nucleotide excision [43, ••44]. Analogous to the NTP-mediated nucleotide excision reaction by HIV-1 RT, that produces dinucleoside tetraphosphate, the excision product by HCV NS5B (RNA polymerase) has ribonucleosides on both ends.…”

“…Analogous to the NTP-mediated nucleotide excision reaction by HIV-1 RT, that produces dinucleoside tetraphosphate, the excision product by HCV NS5B (RNA polymerase) has ribonucleosides on both ends. Using a kinetic study, Jin et al described that HCV polymerase excises chain-terminators and mismatched NMPs from 3′-end of a growing RNA primer [••44]. This result suggests that HCV and other polymerases may be using excision as a general fidelity mechanism.…”