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Mutation rate in hepatitis C virus NS3 protease is not influenced by HIV-1 protease inhibitor therapy

Halfon, Philippea; Bourlière, Marcb; Khiri, Hacènea; Pénaranda, Guillaumec; Martineau, Agnesa; Oulès, Valérieb; Courcambeck, Jérômed; Philibert, Patricka

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doi: 10.1097/QAD.0b013e32830a989b
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Limited analysis by population sequencing has been reported for the selection of isolates with mutations within the NS3 protease that confer resistance to the hepatitis C virus (HCV) protease inhibitors. Little is known about the influence of anti HIV-1 protease inhibitors, through selection pressure, on the HCV protease. We noted, in a cohort of HCV-infected and HIV–HCV-coinfected patients, that the natural strains of the NS3 protease domain related to resistance to HCV-protease inhibitors were well conserved. Anti-HIV-1 protease inhibitors had no influence on the mutation rate in the NS3 protease. This finding could have implications for the future monitoring of HIV–HCV-coinfected patients receiving anti-HCV protease inhibitors.

Encouraged by the stunning success of HIV protease inhibitors in halting the progression of AIDS, researchers turned to HCV protease to treat HCV infection. Since the discovery of the first protease inhibitor, BILN, which was stopped for cardiotoxicity, new protease inhibitors have been developed in human clinical trials [1,2]. Indeed, phase II and III were conducted in HCV patients with SCH5036 (Boceprevir) and VX 950 (Telaprevir) [3,4]. Like HIV-1, HCV persists as a population of multiple, closely-related variants generated by the low-fidelity HCV RNA polymerase. The HCV NS3 protease is a chymotrypsin-like serine-protease responsible for cleavage of the nonstructural proteins of HCV that plays a pivotal role in viral life cycle [5,6]. Limited analysis by population sequencing has been reported for the selection of isolates with mutations within the NS3 protease that confer resistance to the HCV protease inhibitors [7]. Selection of drug-resistant mutants was demonstrated by in-vitro and clinical studies with HCV NS3-4A protease inhibitors [3,4]. It appeared, in in-vitro and in-vivo studies, that mutations V36M, A71T, T72I, P88L, R155Q A156T, D168V, and V170I/M were selected that confer resistance to each protease inhibitor [8–10].

Owing to common routes of transmission (i.e. intravenous drug use and transfusion), one third of the patients infected with the HIV in the USA and Europe are coinfected with HCV [10–12]. From 20% to 40% of HIV–HCV-coinfected patients may achieve a sustained virological response with combined treatment of pegylated interferon plus ribavirin [12–16]. In this therapeutic context, there is an urgent need to develop more specific antiviral drugs associated with a shorter therapy for treating HIV–HCV-coinfected persons [1]. Future therapy for HIV–HCV-coinfected patients will include HCV protease and polymerase inhibitors. Inhibition of wild-type HCV may ‘select’ naturally occurring drug-resistant variants. However, the influence of anti-HIV-1 protease inhibitor, through selection pressure, on the HCV protease is not still established. The aim of the present study was to describe the natural polymorphism of the NS3 sequence in different HCV 1 strains and to compare the diversity of the protease in 33 HCV-monoinfected patients (16 genotype 1a and 17 genotype 1b) and in 17 HIV–HCV-coinfected patients (12 genotype 1a and five genotype 1b) receiving HIV-1 protease inhibitor therapy (Atazanavir boosted by Ritonavir in seven patients, Fosamprenavir boosted by Ritonavir in eight patients, Saquinavir boosted by Ritonavir in two patients). The NS3 protease domain (amino acids 54–197) was amplified by reverse transcriptase-PCR. PCR products were purified and directly sequenced for genotypic and phenotypic analysis of amino acid changes (Fig. 1) [17]. Multiple alignments of nucleotides and deduced amino acid sequences were inferred by Clustal X, version 1.64b. Fisher's exact test was used to compare proportions of mutation at positions 36, 54, 71, 72, 88, 155, 156, 168, and 170. The Wilcoxon rank-sum test was used to estimate clinical and virological differences between HCV-monoinfected patients and HIV–HCV-coinfected patients.

Fig. 1
Fig. 1:
Schematic representation of the sequence analysis of the hepatitis C virus NS3 domain.

The mutation rates observed in the different positions were not different for HCV-infected and HIV–HCV-coinfected patients (19% and 18%, respectively). No differences in aminoacid sequences were found between genotype 1a and genotype 1b patients. Diversity on the protease was more frequently observed in positions 71 and 72; positions 36, 155, 156, 168, and 170 were well conserved regardless of the HCV subtype 1 and the HIV-1 coinfection status. Despite the full 534-bp NS3 protease catalytic domain not being fully analyzed in this study, the limited portion of part of the protease gene analyzed (390-bp for HCV-1a and 421 bp for HCV 1-b) involved the main relevant mutations associated with HCV protease drug resistance.

Sensitive sequencing analysis of HCV protease of patients treated with anti-HIV-1 protease inhibitor demonstrated an absence of selection pressure on the HCV protease. Data should be gathered on the selection of various, so far unknown mutations within the NS3 protease with different resistance levels and increasing frequencies [3,4]. One of the weaknesses of the present study is the low number of patients included in each group.

In conclusion, in this cohort of HCV-infected and HIV–HCV-coinfected patients, the natural strains of the NS3 protease domain related to resistance to HCV-protease inhibitor were well conserved. Anti-HIV protease inhibitor therapy had no influence on the mutation rate in the NS3 protease. This finding could have implications for future monitoring of HIV–HCV-coinfected patients receiving anti-HCV protease inhibitors. Further studies on larger cohorts should confirm this finding.


Part of this paper was presented as an abstract form in the 58th Annual Meeting of the American Association for the Study of Liver Diseases (2–6 November 2007) in Boston.

P.H., M.B., and P.P. designed the study; P.H. wrote the paper; M.B. and V.O. recruited the patients; H.K., A.M., and J.C. performed the reverse transcriptase-PCR analyses; and G.P. performed the statistical analyses. All authors have read and approved the final version of the paper.


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