Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Abstract

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.

FIG. 1.

The HCV genome. The HCV genome contains a single major ORF flanked by untranslated regions (UTR). The 10 proteins encoded within the main ORF are indicated by alternate shading. The ARF protein is encoded in the +1 reading frame within the core region.

FIG. 2.

Number of unique variations per sample by response. The numbers of variations relative to a population consensus that were unique to either the marked or poor responders are shown for the polyprotein (PP) and for each gene within the polyprotein. Statistical significance is shown for genes with P ≤ 0.01. (A) Genotype 1a. (B) Genotype 1b.

FIG. 3.

Sum of Shannon's entropy values by response. The marked- and poor-responder sequences were aligned separately, Shannon's entropy was determined for each position in the two alignments, and the entropy values were summed for each gene. Statistical significance between values for the marked- and poor-responder samples is indicated for genes where P ≤ 0.01. PP, polyprotein. (A) Genotype 1a. (B) Genotype 1b.

FIG. 4.

Average genetic distances by response. The marked- and poor-responder sequences were aligned separately, the genetic distance between each pair of sequences in the alignments was calculated using the p-distance algorithm, and the average genetic distance for each protein in the alignment was plotted. The statistical significance of the difference between the groups is indicated for those genes where P ≤ 0.01. (A) Genotype 1. (B) Genotype 1b.

FIG. 5.

Numbers of unique variations per sample for all three response classes. The numbers of variations relative to a population consensus that were unique to the marked, intermediate, or poor-responder sequences are shown for the full genotype 1a and 1b polyproteins.

FIG. 6.

Numbers of unique variations per sample by race. The numbers of variations relative to a population consensus that were unique to either the AA or CA sequences are shown for the polyprotein (PP) and for each gene within the polyprotein. Statistical significance is shown for genes with P ≤ 0.01. (A) Genotype 1a. (B) Genotype 1b.

FIG. 7.

Sum of Shannon's entropy values by race. The AA and CA sequences were aligned separately, Shannon's entropy was determined for each position in the two alignments, and the entropy values were summed for each gene. Statistical significance between values for the AA and CA samples is indicated for genes where P ≤ 0.01. (A) Genotype 1a. (B) Genotype 1b.

FIG. 8.

Average genetic distances by race. The AA and CA sequences were aligned separately, the genetic distance between each pair of sequences in each of the two alignments was calculated using the p-distance algorithm, and the average genetic distance for each protein in the alignment was plotted. The significance of the difference between the groups was determined using an independent sample t test and is indicated for those genes where P ≤ 0.01. (A) Genotype 1. (B) Genotype 1b.

FIG. 9.

Effect of race on genetic variation associated with response to therapy. Variations relative to a population consensus that were unique to the marked- and poor-responder samples were divided by race, and a log-likelihood test was performed to determine whether race affected the diversity associated with response to therapy. Statistical significance is shown for domains with P ≤ 0.01; the upper P value is for the log-likelihood interaction of race and response, and the lower P value is for the Poisson analysis of the indicated response groups. M, marked; P, poor; PP, polyprotein. (A) Genotype 1a. (B) Genotype 1b.

FIG. 10.

Numbers of unique variations in the nonepitope regions by response. Variations relative to the population consensus sequence that were not in any known or predicted T-cell epitope were compared in the marked- and poor-responder samples. Statistical significance is indicated where P ≤ 0.01. PP, polyprotein. (A) Genotype 1a. (B) Genotype 1b.

FIG. 11.

Numbers of variations in NS3 protease and helicase domains by response. Variations relative to a population consensus sequence in the NS3 protease and helicase domains were compared in the marked- and poor-responder samples. Statistical significance is shown for domains with P ≤ 0.01. (A) All variations in genotype 1a NS3 domains. (B) Variations unique to the marked- or poor-responder sequences for genotype 1a NS3 domains. (C) All variations in genotype 1b NS3 domains. (D) Variations unique to the marked- or poor-responder sequences for genotype 1b NS3 domains.

FIG. 12.

Variations in the genotype 1a NS5A ISDR/PKR binding domain. Sequences encompassing the ISDR/PKR binding site in NS5A from CA and AA marked (M) and poor (P) responders were aligned relative to the genotype 1a population consensus sequence (Ref). Positions where the Virahep-C sequences differ from the reference sequence are indicated. Numbers at the top indicate amino acid positions in NS5A.