Effects of Mutagenic and Chain-Terminating Nucleotide Analogs on Enzymes Isolated from Hepatitis C Virus Strains of Various Genotypes

Abstract

The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors ( , only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.Hepatitis C virus (HCV) infects more than 170 million people worldwide, causing chronic hepatitis, cirrhosis, hepatocellular carcinoma, and/or liver failure (12). Clinical HCV isolates are categorized into different genotypes that display up to 30% sequence variation. Response rates to all interferon-based HCV therapies are genotype dependent, with patients infected with HCV genotype 1 having about one-half the chance of a sustained virological response as patients infected with other genotypes (37). Current HCV therapies combine pegylated alpha interferon and ribavirin (1-␤-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). A number of viral factors have been proposed to contribute to the different response rates to interferon (reviewed in reference 59), but less is known about the effects of viral variation on sensitivity to ribavirin. Here, we employ biochemical methods to examine the simple hypothesis that as a nucleoside analog, ribavirin may affect the enzymes encoded by HCV, and that variation among the enzymes from different genotypes could affect ribavirin response rates. We also examine whether HCV genetic variation influences the sensitivities of HCV enzymes to other nucleoside analogs similar to those currently in development as HCV-specific drugs.The ϳ9,600-nucleotide HCV genome harbors a single open reading frame that encodes a polyprotein, which is processed into 10 distinct proteins by both host and v...

The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5′-Triphosphate towards Influenza A Virus Polymerase

“…We used 3′dGTP as a positive control for polymerase inhibition because it is an obligate chain terminator [23] (Fig. 1A).…”

Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

“…The truncated C-terminal His-tagged NS3 proteins lacking the protease domain (i.e. NS3h) were purified as described before: NS3h_1a(JFH1), NS3h_1b(J4), NS3h_2a(J6) (14), NS3h_ 1b(con1) (15), NS3h_2a(JFH1) (8), NS3h_R393A (16), NS3h_E493Q (17). An open reading frame expressing NS3h_S231A was generated for this study from a plasmid expressing NS3h_1b(con1) by Mutagenex Inc. (Hillsborough, NJ).…”

PSI-7851, a Pronucleotide of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methyluridine Monophosphate, Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

“…The digested PCR product was ligated into the pET23a vector (EMD Chemicals Inc., Darmstadt, Germany) to generate a C-terminal His-tagged expression construct. 2a_JFH1 NS5B⌬21 was expressed in Rosetta (DE3) Escherichia coli and purified as described previously (13). Genotype 3a and 4a NS5B polymerases were constructed from human serum samples containing genotype 3a or 4a HCV (SeraCare Life Sciences, Milford, MA), from which viral RNA was isolated using the QIAamp MinElute virus spin kit (Qiagen, Valencia, CA).…”

“…Two major drawbacks associated with NNIs are that the activity appears to vary significantly among different HCV genotypes and even subtypes (15,33) and that there is a relatively low barrier for resistance as evidenced by the numerous naturally occurring resistant variants reported in the literature (18). In contrast, nucleoside analogs are similarly active across HCV genotypes (13,15,33) and have a higher barrier of resistance compared to the NNIs and NS3 protease inhibitors (36). To date only two amino acid changes within the NS5B polymerase that confer resistance to nucleoside inhibitors have been identified: S96T and S282T (1,29).…”