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

Abstract

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2-C-methyl CTP (2-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2-modified nucleotide analogues. 2-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2-Omethyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5-to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.Hepatitis C virus (HCV) infection is the most common blood-borne infection and a major cause of chronic liver disease in developed countries. More than 170 million individuals in the world are infected with HCV, making these individuals at risk of developing liver cirrhosis and hepatocellular carcinoma. Persistent HCV infection can be controlled by antiviral therapy (30, 40) or even eradicated from infected patients (43).Current antiviral therapies rely on the combination of pegylated alpha interferon and the nucleoside analogue ribavirin (16,18,29). Less than 50% of treated patients respond when they are infected with the most prevalent HCV genotype, i.e., genotype 1 (23). The HCV RNA-dependent RNA polymerase (RdRp), the NS5B protein, plays an essential role in the replication of the viral genome and is thus an attractive target for the development of new antiviral drugs (36). Intensive drugscreening programs led to the discovery of two classes of HCV NS5B inhibitors, namely, nonnucleoside inhibitors (NNIs) and nucleoside inhibitors (NIs). NNIs have been described to bind to one of the three allosteric sites present at the NS5B surface (for a review, see reference 11). All of the NNIs are noncompetitive inhibitors relative to nucleoside triphosphate (NTP) incorporation and target the alloenzyme free of substrate. They are inactive when the enzyme has entered into the processive elongation phase (3,17,31,45,46) of in vitro RdRp reactions or when NS5B is c...

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

“…It is also interesting to note that position 316 is under high selective pressure in this subtype, as shown by a high dN/dS ratio (equal to 16). The high frequency of mutations detected in our study sample is remarkable and suggests that their occurrence is sufficiently spread out, as previously reported (Horiike et al 1999, Qin et al 2001, Hamano et al 2005, Dutartre et al 2006, Lutchman et al 2007, Kuntzen et al 2008, McCown et al 2009, rydberg et al 2009). rIB selects for non-synonymous mutations, increasing sensitivity to IFN and leading to SVr status.…”

“…Two other codons (282 and 316) implicated with resistance to new NS5B inhibitors were also within the NS5B fragment sequenced in this study (Dutartre et al 2006, Shi et al 2008, McCown et al 2009). Codon 282 was implicated with mutations related to 2me-cytosine resistance (mutation S282T) and all isolates analysed coded for serine in this position (Dutartre et al 2006).…”

“…Codon 282 was implicated with mutations related to 2me-cytosine resistance (mutation S282T) and all isolates analysed coded for serine in this position (Dutartre et al 2006). The C316N mutation was previously related to a new non-nucleoside compound (HCV796; ViroPharma, Exton, Pennsylvania and Wyeth research, Philadelphia, Pennsylvania), but eight out of 33 (24%) patients with subtype 1b already carried this primary mutation despite never having been exposed to this new agent (Table I).…”

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

“…Together, these findings suggest that the excised chain terminator can be reincorporated in the absence of the competing natural counterpart that is only present in the excision/rescue reaction. At the same time, these data help to explain why pyrophosphorolysis is sometimes difficult to observe when the particular sequence context diminishes the reaction per se (11).…”

“…In a single-nucleotide competition experiment, the mutant enzyme showed increases in IC 50 s of between 9.5-fold and Ͼ24-fold for 2Ј-C-Me-CTP and 2Ј-CMe-ATP, respectively ( Table 2). Increases in K m values suggest that the mutated enzyme displays decreased inhibitor binding (11). Formation of DEC.…”