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. 1999 Apr;73(4):2938-46.
doi: 10.1128/JVI.73.4.2938-2946.1999.

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy

S C Ray  1 , Y M Wang, O Laeyendecker, J R Ticehurst, S A Villano, D L Thomas
Affiliations

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy

S C Ray et al. J Virol. 1999 Apr.

Abstract

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.

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Figures

FIG. 1
FIG. 1
Diagram depicting the studied portion of the HCV genome and locations of the PCR primers (arrowheads) used in this study. Positions are based on the work of Choo et al. (12). 5′NCR and 3′NCR, 5′ and 3′ noncoding region, respectively.
FIG. 2
FIG. 2
Alignment of inferred amino acid sequences for the majority sequences from each subject. In the first column, an alphabetical label is given for each subject, while in the second column, C indicates clearance of viremia and P indicates persistence. Periods indicate identity to the amino acid at that position in the first sequence. Positions of the Core and E1 and E2 regions are indicated above the alignment, whereas that of HVR1 is indicated below the alignment at the N terminus of E2. Boxes indicate predicted N-linked glycosylation sites. Cysteine residues in the first sequence are underlined.
FIG. 3
FIG. 3
Comparison of the frequencies of amino acids in consensus sequences for the 5 case subjects (group showing clearance) and the 10 control subjects (group showing persistence of viremia). A subscript indicates the number of sequences having that residue at that position. Sites identified by using signature pattern analysis are displayed; for sites not shown, the most frequently observed amino acids (aa) did not differ between case subjects and control subjects. Positions are based on the work of Choo et al. (12). Also shown are the amino acids in these positions for 58 GenBank sequences spanning the same region.
FIG. 4
FIG. 4
Virologic correlates of outcome. (A) Clonotype ratio, calculated as the ratio of the number of clonotypes detected to the number of cloned DNAs examined (Table 2), versus outcome. (B) E1 dN/dS ratio versus outcome. For each subject, all pairwise nonsynonymous and synonymous distances were calculated for the E1-coding region. These distances were averaged, and the dN/dS ratio was then calculated. Horizontal lines represent medians.
FIG. 5
FIG. 5
Variability plots of the envelope region. For each subject, the intrasample dN/dS ratio (A), dN value (B), dS value (C), or Δd value (dN − dS) (D) was calculated for overlapping windows of 20 amino acids (aa; 60 nucleotides), sliding in increments of 1 amino acid across E1 and the first 119 amino acids of E2. The mean values for each group (clearance or persistence) were then plotted. Positions are based on the work of Choo et al. (12).
FIG. 6
FIG. 6
Ratio of nonsynonymous to synonymous distances versus clonotype ratio. Values from Fig. 4A and B were plotted on the same graph, and a box (dotted line) is drawn around the points representing values for the case subjects (with clearance of viremia).
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