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There are limited data on diversity within the hepatitis C virus polymerase (NS5B). In concordance with its key functional role during the life cycle, NS5B intrapatient variability was low. Moreover, differences between NS5B nonsynonymous (dN) and synonymous (dS) mutation rates (dN − dS) were positively correlated with CD4 cell count, while nonsynonymous mutations were strongly correlated with reduced replication in vivo.
The NS5B protein of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase that lacks a proofreading mechanism, resulting in a population of distinct but closely related viral variants, termed viral quasispecies, within an infected individual (16). The advent of potent NS5B inhibitors mandated a focus on the identification and monitoring of putative NS5B resistance mutations. HCV quasispecies diversity is an important predictor of liver disease progression as well as HCV treatment outcome (7, 13-15, 18). Most of these studies have focused on structural genomic regions; thus, limited data are available regarding nonstructural regions. To address this issue, we assessed serum NS5B variability in chronic HCV infection. (These data were presented at the 4th International Workshop on HIV and Hepatitis Co-Infection held in Madrid, Spain, in June 2008.)
From 1993 to 2000, a prospective natural history study of HIV infection, the HER Study, was conducted with U.S. women (17). Women were included in the current study if they were (i) HCV antibody positive, (ii) from the Rhode Island study site, and (iii) had available serum samples for analysis. All participants provided informed consent prior to sample and clinical data collection. RNA extractions and amplifications of a 384-bp region of NS5B were performed as described previously (4). Gel-purified PCR products were ligated into the pGEM-T Easy vector, and a median of 10 plasmids per sample was sequenced. We and others have shown that analysis of ~10 sequences accurately and reproducibly represents intrapatient quasispecies diversity (1, 20). All alignments were performed using Clustal X and compared to database references to determine the HCV genotype. Intrapatient genetic distances, Shannon entropy, and differences between nonsynonymous (dN) and synonymous (dS) rates (dN − dS) were calculated as described elsewhere (1). Codons under positive or negative selection were detected via the fixed effects likelihood (FEL) and random effects likelihood (REL) methods as implemented in the Datamonkey program (12). P values of <0.05 were considered statistically significant.
A randomly selected subset of genotype 1-infected women that included 25 with HIV/HCV coinfection and 4 with HCV monoinfection were analyzed. Mean aspartate transaminase (AST) and alanine aminotransferase (ALT) levels were 58.5 U/ml and 51.9 U/ml, respectively. For the HIV/HCV-coinfected women, the median CD4 cell count was 358.7 cells/ml, and 23 (92%) had detectable plasma HIV viral loads. Of 22 HIV/HCV-coinfected women with available data, 13 (59%) were receiving antiretroviral therapy at the time of serum collection. Twenty-six women were infected with HCV genotype 1a, and 3 were infected with genotype 1b. Among the partial NS5B consensus sequences generated, 21 of 114 (18.4%) amino acid positions analyzed were variable (Fig. (Fig.1).1). The NS5B motifs A to D were conserved at nearly all amino acid positions, including the GDD active site. S282T and C316Y resistance mutations were not observed in any serum samples, although we have previously reported the persistence of the C316Y mutation in a treatment-naïve HCV-monoinfected woman from the same cohort (4). We further evaluated multiple measures of NS5B intrapatient variability. The median intrapatient genetic distance was 0.90% (range, 0.30% to 1.50%). These values are lower than we reported previously for hypervariable region 1 (HVR1) but higher than genetic distance measures for the 5′-untranslated region (1). Median intrapatient entropy was 0.83 (range, 0.53 to 1.00). The median intrapatient dN − dS value for NS5B was −0.011 (range, −0.035 to 0.005), indicating negative or purifying immune selection pressures have preserved this functionally constrained genomic region. While it was not our intent to compare NS5B diversity in the presence/absence of HIV coinfection, we did observe a higher median genetic distance for the HIV/HCV-coinfected women than for the HCV-monoinfected women (0.8% versus 0.4%; P = 0.002), even with the small population size. Site-specific codon selection was examined in each patient as summarized in Table Table1.1. Using the FEL method, there were no positively selected amino acid sites in NS5B among the 29 women examined; however, there were 14 negatively selected amino acid sites identified in 9 women. Three of these amino acids—2730, 2747, and 2769 of the region analyzed—were identified in more than one woman. Using the REL method, 2 amino acids—2807 and 2818—were found to be under positive selection pressure, while 48 amino acids in seven women (2 to 13 sites per individual) were under negative selection pressure. Four of these amino acids—2747, 2789, 2800, and 2817—were identified in at least two women, while amino acid 2769 was identified in three women. We also analyzed potential correlations among various clinical parameters and measurements of quasispecies diversity (Table (Table2).2). Median intrapatient NS5B dN − dS values trended toward a positive correlation with CD4 cell count (Spearman r, 0.387; P = 0.056) but were negatively correlated with HCV RNA levels (r, −0.492; P = 0.007). NS5B genetic distance, entropy, and dN − dS were not correlated with ALT, AST, or HIV viral load. Similar results were obtained when the study population was restricted to those with HIV/HCV coinfection.
Few data are available for nonstructural genomic regions, despite their potential as therapeutic targets to reduce viral replication. Our data support the concept that viral load is influenced by variability within the polymerase gene. This observation is supported by a number of in vitro studies that have identified key positions within NS5B that can dramatically affect HCV replication (9, 10). Moreover, a correlation has been demonstrated between the number of amino acid substitutions in NS5B and replicative capacity (6, 8), as well as HCV treatment outcome (5). Thus, increased mutations within NS5B may have a deleterious effect on viral replication both in vitro and in vivo. In the current study, intrapatient NS5B variability was detected, although variability was not as high as that observed in HVR1 from the same individuals (unpublished observations). The existence of multiple unique NS5B variants within an infected individual permits rapid viral adaptation to immunologic and antiviral selection pressures and the cellular microenvironment. We identified a number of amino acid positions that were under negative selection, but only two amino acids were found to be under positive selection. Moreover, among genotype 1-infected women, the median dN − dS value for NS5B was <0, suggesting that negative immune selection pressures and/or strong functional constraints on enzymatic activity limit NS5B variability compared to other more variable genomic regions. Nonetheless, intrapatient dN − dS values were positively correlated with CD4 cell count but negatively correlated with HCV RNA levels, suggesting that nonsynonymous mutations within NS5B could impact viral replication. In contrast, NS5B variability was not associated with HIV viral load, although the median NS5B genetic distance was higher for HIV/HCV-coinfected women than HCV-monoinfected women. This finding is particularly interesting given that HCV RNA levels are significantly elevated during HIV/HCV coinfection (19, 21) and HIV coinfection is associated with lower HCV treatment response (2, 11). Thus, HIV may influence NS5B variability, although this intriguing possibility requires a longitudinal analysis of NS5B diversity.
Our study has several potential limitations, including the modest population size, analysis of partial NS5B sequences, and the inclusion of relatively few HCV-monoinfected controls. The analysis included only HCV genotype 1, and the relevance of our findings to non-1 genotypes is unknown. While our data may be influenced by PCR-generated mutations, the various measures of quasispecies diversity reported here suggest that NS5B diversity was in excess of the Taq polymerase error rate (3). Future studies aimed at functional characterization of unique NS5B sequences identified in vivo will also provide data on how NS5B variability impacts replicative fitness. In summary, our data demonstrated a link between NS5B viral diversity and viral load that could impact targeted drug therapy. Longitudinal analysis of the cohort with relation to disease and treatment outcome is warranted.
Consensus NS5B sequences have been submitted to GenBank under accession numbers GU131368 to GU131396.
We thank the HER Study staff and participants. The HER Study group consists of Robert S. Klein, Ellie Schoenbaum, Julia Arnsten, Robert D. Burk, Penelope Demas, and Andrea Howard from Montefiore Medical Center and the Albert Einstein College of Medicine; Paula Schuman, Jack Sobel, Suzanne Ohmit, William Brown, Michael Long, Wayne Lancaster, and Jose Vazquez from the Wayne State University School of Medicine; Anne Rompalo, David Vlahov, and David Celentano from the Johns Hopkins University School of Medicine; Charles Carpenter, Kenneth Mayer, Susan Cu-Uvin, Timothy Flanigan, Joseph Hogan, Valerie Stone, Karen Tashima, and Josiah Rich from the Brown University School of Medicine; Ann Duerr, Lytt I. Gardner, Chad Heilig, Scott D. Holmberg, Denise J. Jamieson, Janet S. Moore, Ruby M. Phelps, Dawn K. Smith, and Dora Warren from the Centers for Disease Control and Prevention; and Katherine Davenny from the National Institute on Drug Abuse.
This work was supported by the Partners/Fenway/Shattuck Center for AIDS Research (NIH P30-AI42851); the National Institute on Drug Abuse (R21 DA022148-01); the Life Span/Tufts/Brown Center for AIDS Research (NIH P30 AI42853); the Japanese Ministry of Education, Culture, Sports, Science, and Technology (JSPS KAKENHI 21590848); and the Centers for Disease Control and Prevention (U64/CCU106795 to fund data collection at Brown University).
Published ahead of print on 1 September 2010.