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

Abstract

Nonobligate chain terminators, such as 2-C-methylated nucleotides, block RNA synthesis by the RNAdependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.Hepatitis C virus (HCV) infection is a serious public health concern that affects 170 million people worldwide (33, 42). Among those infected, approximately 20 to 30% develop severe liver disease, such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (2). The combined use of the nucleoside analogue ribavirin and pegylated alpha interferon is the current treatment standard; however, success in treatment depends largely on the viral genotype, and this drug combination has also been associated with severe side effects (30,43). Thus, the development of novel, more potent, specific drugs is urgent.HCV belongs to the Flaviviridae family. The HCV RNA genome consists of approximately 10 kb, encoding a polyprotein which is processed into several smaller polypeptides, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). 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. Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanc...

General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2′-Modified Nucleotide Analogues

“…No pyrophosphorolysis could be observed in our experimental system with either WT or S282T NS5B, probably because of specific template requirements for this particular mechanism (14). Nevertheless, it was shown that S282T NS5B is able to excise chain-terminator analogues to the same extent as WT NS5B (14). Therefore, S282T mutant-mediated re- Tables 1 and 3 for comparative incorporation during initiation and from Tables 2 and 4 sistance to 2Ј-C-Me-NTP is only due to K m -based discrimination (Table 2).…”

“…It was recently reported that HCV and bovine viral diarrhea virus polymerase resistance to nucleotide analogues could also be the result of pyrophosphorolysis leading to the excision of the analogue after its incorporation into the nascent RNA (10,14). No pyrophosphorolysis could be observed in our experimental system with either WT or S282T NS5B, probably because of specific template requirements for this particular mechanism (14). Nevertheless, it was shown that S282T NS5B is able to excise chain-terminator analogues to the same extent as WT NS5B (14).…”

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

“…The coding sequence for HCV NS5B (genotype 1b, BK strain) was cloned into the bacterial expression vector pet21b. The hydrophobic region at the C terminus was removed (⌬21), and a hexa-histidine tag was introduced to facilitate protein purification (20,36). Mutant enzymes were generated via site-directed mutagenesis using the Stratagene QuikChange kit according to the manufacturer's protocol.…”

Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

“…The antiviral activity of 5-fluorocytidine against RNA and DNA viruses has been previously reported (29), as has the anti-HIV-1 activity of the reduced form of 5-fluorocytidine, 2=-deoxy-5-fluorocytidine (30), and that of the derivative 2=,3=-dideoxy-beta-L-5-fluorocytidine (30). Previous reports have demonstrated the antiviral activity of 2=-C-methylcytidine as a delayed chain terminator against RNA viruses, such as hepatitis C virus (HCV) and footand-mouth disease virus (FMDV) (31)(32)(33). Our observations of anti-HIV-1 activity associated with 2=-C-methylcytidine are the first to be reported.…”