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Hepatitis C virus (HCV) non-nucleoside inhibitors (NNIs) target the viral RNA-dependent RNA polymerase encoded by the NS5B gene. Several NNIs share a similar allosteric binding site, and their antiviral efficacy is attenuated by a cysteine-to-tyrosine mutation at amino acid 316 (C316Y). In the current study, we assessed NS5B resistance mutations in treatment-naive individuals from a prospective natural history study of viral infections in women.
Partial NS5B sequences from HCV-positive women were amplified by RT–PCR. Additionally, subcloning was performed to evaluate intrapatient variability in selected samples.
HCV NS5B genotypes were 45 genotype 1a (57.0%), 11 genotype 1b (13.9%), 5 genotype 2a (6.3%), 3 genotype 2b (3.8%), 9 genotype 3a (11.4%) and 6 genotype 4a (7.6%). One HCV genotype 1a-infected patient was found to have the C316Y mutation (1.3%). Clonal analysis further revealed that all NS5B sequences from this individual—representing three serum samples collected 4 years apart—contained the C316Y mutation. In contrast, the S282T resistance mutation was not found in any samples.
The C316Y polymerase resistance mutation was found in 1.3% of samples from HCV-infected women. The presence of this mutation over time suggests significant replicative fitness of this variant and has implications for development of new specifically targeted antiviral therapies against HCV (STAT-C) targeting this region.
An estimated 170 million people are infected with chronic hepatitis C virus (HCV) worldwide. The viral NS5B gene encodes an RNA-dependent, RNA polymerase that lacks a proofreading mechanism, thus generating extensive viral diversity. This leads to development of viral quasispecies—distinct viral variants within an individual—as well as multiple HCV genotypes at the population level.
Specifically targeted antiviral therapies against HCV (STAT-C) are in active development and represent the newest advances in HCV treatment. STAT-C agents include protease inhibitors, nucleoside inhibitors and non-nucleoside inhibitors (NNIs), as well as less explored targets. A subset of NNIs binds to the palm domain near the polymerase active site. Among these, experimental agents such as GS-9190 and HCV-796 demonstrate antiviral activity in human clinical trials, while A-837093 has shown promise in animal models.1–3 STAT-C monotherapy effectively reduces viral load shortly after treatment initiation. However, the existence of low level, pre-existing resistance mutations permits the rapid emergence of drug resistance and treatment failure during monotherapy.1,3,4 As originally described by Villano et al.,4 the most frequently observed mutation associated with HCV-796 resistance is the cysteine-to-tyrosine signature mutation at position 316 of NS5B (C316Y). C316Y is associated with a 166-fold reduction in susceptibility to HCV-796 in vitro. Additionally, the cysteine-to-asparagine polymorphism (C316N) confers a modest 26-fold reduction in susceptibility and is relatively common in genotype 1b-infected individuals but is not considered a drug resistance mutation.5 However, the prevalence of the C316Y mutation in untreated individuals has not been adequately explored outside of the clinical trial setting. We analysed data from the HIV Epidemiologic Research (HER) Study—a prospective natural history study of HIV infection in women conducted from 1993 to 19996—to assess NS5B resistance mutations in treatment-naive individuals.
A total of 871 HIV-infected women and 439 demographically matched, uninfected women were recruited from four US sites into the HER Study. By study design, approximately half of the women reported having used injection drugs at least once since 1985, while the other half reported only sexual risk behaviours. All participants provided informed consent prior to sample and clinical data collection. Institutional review boards approved the study at each site and at the CDC. The overall prevalence of HCV was 56.5%.7 For the current analysis, HER Study subjects were included if they were: (i) HCV antibody positive at baseline; (ii) from the Rhode Island site; and (iii) had serum samples available for additional analysis. Both HIV-positive and HIV-negative subjects were included. Of the 327 women enrolled at the Rhode Island study site, 169 were HCV seropositive and had detectable HCV RNA. A total of 129 of these samples were available for the current analysis, and NS5B could be amplified by direct sequencing for 79.
Viral RNA was extracted from serum using the QIAamp Viral RNA kit (Qiagen). A partial segment of the NS5B gene was amplified using a One-Step RT-PCR kit (Qiagen) and the primers PR3: TAT gAY ACC CgC TgY TTT gAC TC (nucleotides 8256–8278 relative to the start of H77) and PR4: gCN gAR TAY CTV gTC ATA gCC TC (nucleotides 8644–8622). Reaction conditions were 30 min at 50°C, 15 min at 95°C, followed by 5 cycles of 30 s at 93°C, 45 s at 60°C and 60 s at 72°C, followed by 35 cycles of 30 s at 93°C, 45 s at 60°C to 50°C and 60 s at 72°C, followed by a 5 min final elongation step at 72°C. First-round PCR products were then amplified by hemi-nested PCR with the primers PR3 and PR5: gCT AgT CAT AgC CTC CgT (nucleotides 8636–8619) using the GeneAmp PCR kit (Applied Biosystems). Reaction conditions were 2 min at 95°C, followed by 36 cycles of 30 s at 95°C, 45 s at 55°C and 1 min at 72°C, followed by a final elongation step for 10 min at 72°C. PCR products were gel purified and sequenced directly using dye terminator chemistry. To confirm these sequence results, multiple clones per time point were ligated into the pGEM-T Easy Vector (Promega) and sequenced for a subset of individuals.
Sequences corresponding to amino acids 227–344 of the polymerase were aligned using the neighbour-joining method in the ClustalX software. The GenBank database references used to confirm HCV genotype included 1a (AF009606, AJ278830, AF511948), 1b (AJ000009, D10934), 1c (D14853), 2a (AB047639), 2b (AB030907), 2c (D50409), 3a (D28917), 3k (D63821), 4a (Y11604) and 6a (Y12083). The statistical robustness and reliability of the branching order within the phylogenetic tree were confirmed by bootstrap analysis using 100 replicates. The region of NS5B amplified included the resistance mutations S282T8 and C316Y.5 These mutations were assessed in consensus, as well as clonal sequences, when available.
The NS5B region was amplified and sequenced directly from 79 patient serum samples. Demographic and clinical details of this cohort are provided elsewhere.9 Briefly, the HCV genotype distribution included 45 genotype 1a (57.0%), 11 genotype 1b (13.9%), 5 genotype 2a (6.3%), 3 genotype 2b (3.8%), 9 genotype 3a (11.4%) and 6 genotype 4a (7.6%). Amino acid alignment did not reveal the presence of the other major resistance mutation—S282T—or any known compensatory mutations in the region analysed for any of the 79 women. In contrast, one individual possessed the C316Y mutation in the consensus sequence from a serum sample collected on 17 March 1994. This patient was a 37-year-old, HIV-negative, white female with a history of injection drug use. Direct sequencing of NS5B demonstrated that she was infected with HCV genotype 1a. Clonal analysis of multiple viral variants from the initial time point, as well as two additional time points collected on 7 September 1994 and 17 August 1998, also revealed the presence of C316Y as the dominant amino acid at this position (20 of 20 clones).
Our understanding of HCV resistance mutations reflects lessons previously learned from the HIV literature. For instance, drug-resistant viruses are generally less fit than wild-type viruses, though high degrees of viral fitness with certain variants have been observed.10 Moreover, drug-resistant viruses are often present at very low frequencies in treatment-naive individuals, yet rapidly emerge during the course of antiviral therapy.11 Using a genotype 1b replicon system, others have demonstrated that the C316Y mutation confers a 30% reduction in viral fitness compared with the wild-type in vitro.12 However, resistance may be more complicated in vivo, as a chimpanzee infected with HCV 1b maintained the C316Y mutation, as well as a glycine-to-aspartic acid mutation at position 554 (G554D), even after the cessation of therapy.3 Thus, compensatory mutations may also play a role in maintaining certain drug resistance mutations in vivo even after treatment discontinuation. Likewise, HCV RNA levels of naturally occurring NS3/NS5B drug-resistant isolates from untreated individuals are equivalent to those of non-resistant/wild-type virus isolates.13 Thus, these naturally occurring mutations probably confer a selective advantage in vivo, although the existence of compensatory mutations will require further longitudinal analysis in large population-based studies.
In the current analysis, positions 217–347 of NS5B were examined, covering known resistance mutations at residues 282 and 316. The incidence of the C316Y mutation was 1 in 79 patients (1.3%). Importantly, we were able to detect the C316Y mutation by direct sequencing; thus, C316Y represents the dominant amino acid present in the viral quasispecies of this subject. An additional 149 representative sequences from the HCV Sequence Database were also reviewed for the presence of the C316Y mutation; however, no other sequences contained the C316Y mutation (Table 1). Interestingly, a recent analysis of 507 HCV treatment-naive patients reported predominant NS5B resistance mutations in 10 individuals at position 423 and one individual at position 415 (2.8% combined); however, no C316Y resistance mutations were reported.13 C316 is highly conserved in genotype 1a, but polymorphic in genotype 1b.1,5,13 Others have noted that the baseline frequency of HCV drug resistance mutations is 5.0%–8.6%, which is sufficiently high to justify resistance testing, assuming similar costs and response rates of antiretrovirals against HIV.13
Our finding contributes to emerging evidence suggesting that NNI resistance mutations can be maintained as the dominant sequence over the course of several years in untreated individuals, thereby potentially limiting the use of particular NNIs in a subset of individuals. Further longitudinal evaluation of resistance profiles in treatment-naive individuals may better define the demographic and virological predictors of STAT-C outcome.
This work was supported by an NIDA R21 (DA022148) award to J. T. B. and an NIDDK K24 (DK 070528) award to K. E. S. Data collection at Brown University was funded by the CDC cooperative agreement U64/CCU106795.
None to declare.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.
This work was presented at the Thirteenth International Symposium on Viral Hepatitis and Liver Diseases, Washington, DC, 2009 (Abstract P-121).
We would like to thank the HER Study staff and participants. The HER Study group consists of: Robert S. Klein, MD, Ellie Schoenbaum, MD, Julia Arnsten, MD, MPH, Robert D. Burk, MD, Penelope Demas, PhD, and Andrea Howard, MD, MSc, from Montefiore Medical Center and the Albert Einstein College of Medicine; Paula Schuman, MD, Jack Sobel, MD, Suzanne Ohmit, PhD, William Brown, PhD, Michael Long, PhD, Wayne Lancaster, PhD, and Jose Vazquez, MD, from the Wayne State University School of Medicine; Anne Rompalo, MD, David Vlahov, PhD, and David Celentano, PhD, from the Johns Hopkins University School of Medicine; Charles Carpenter, MD, Kenneth Mayer, MD, Susan Cu-Uvin, MD, Timothy Flanigan, MD, Joseph Hogan, ScD, Valerie Stone, MD, Karen Tashima, MD, and Josiah Rich, MD, from the Brown University School of Medicine; Ann Duerr, MD, PhD, Lytt I. Gardner, PhD, Chad Heilig, PhD, Scott D. Holmberg, MD, Denise J. Jamieson, MD, MPH, Janet S. Moore, PhD, Ruby M. Phelps, BS, Dawn K. Smith, MD, MPH, and Dora Warren, PhD, from the CDC; and Katherine Davenny, MPH, from the National Institute of Drug Abuse.