Evolution of hepatitis C virus NS5A region in breakthrough patients during pegylated interferon and ribavirin therapy*

Abstract

Investigating the evolution of the hepatitis C viral (HCV) genome in the small number of patients that experience viral breakthrough might shed light on the problem of resistance to interferon therapy. Within the HCV genome, sequence diversity of the viral nonstructural 5A protein-coding region (NS5A) has been linked to interferon responsiveness. We analysed the temporal sequence changes within NS5A in genotype 1a patients: 6 breakthrough (BT), 12 sustained virologic responders (SVR) and 12 non-responders (NR), all of whom had received full dose peg-interferon and ribavirin therapy. The entire NS5A region was amplified by reverse transcription (RT)-PCR followed by direct sequencing of serum samples from baseline and three on-treatment time points for each group. Comparing baseline sequences with week 12 and later time points, BT patients resembled SVR patients in having a higher number of amino acid substitutions at week 12 than NR patients; however, the number of amino acid substitutions in this group decreased at and after BT. Substitutions were focused in the V3 and flanking regions in BT patients but not in SVR patients. The high number of substitutions in NS5A in both BT and SVR groups suggests that selective pressure is associated with viral response to therapy. Our results provide evidence that amino acid substitutions within the NS5A coding region may reflect a host response that drives selective pressure for viral adaptation.

Figure 1

Serum HCV RNA levels over time in BT(1a), NR(1b) and SVR(1c) patients. Serum HCV RNA levels in all SVR and BT patients were undetectable by definition at week 20. Lower limit of detection was 100 IU/mL.

Figure 1

Serum HCV RNA levels over time in BT(1a), NR(1b) and SVR(1c) patients. Serum HCV RNA levels in all SVR and BT patients were undetectable by definition at week 20. Lower limit of detection was 100 IU/mL.

Figure 2

Phylogenetic analysis of sequences from non-responders (●), SVR (■) and BT (▲) patients. In the phylogenetic tree of baseline nucleotide sequences, there were no significant clusters of sequences according to response. When nucleotide sequences from later time points (black: Baseline, blue: wk 4, green: wk 12, red: BT (or wk 24 in NR), grey: post-BT) were added, the later time point sequences diverged slightly from baseline sequences, but still clustered around the patient profile, rather than response to therapy.

Figure 3

The number of amino acid substitutions at different time points. The lines represent the median and 25th and 75th percentile value in BT, SVR and NR patients. Blue line represents BT patients; dark red line SVR patients and black line NR patients. BT patients demonstrated the greatest number of amino acid substitutions at week 12, similar to that seen with SVR patients, while NR patients demonstrated little or no genetic drift over time.

Figure 4

The distribution of amino acid substitutions across the NS5A region in the period between week 4 and week 12 in SVR (Panel A), BT (Panel B) and NR (Panel C) patients. Each line represents one patient, with vertical lines indicating amino acid substitutions between weeks 4 and 12. Most substitutions occurred in the carboxyl terminal region around V3. There were more amino acid substitutions overall in the SVR and BT patients than were observed in the NR patients. Six NR patients, listed as 7–12, demonstrated no mutations whatsoever in this region.

Figure 4

The distribution of amino acid substitutions across the NS5A region in the period between week 4 and week 12 in SVR (Panel A), BT (Panel B) and NR (Panel C) patients. Each line represents one patient, with vertical lines indicating amino acid substitutions between weeks 4 and 12. Most substitutions occurred in the carboxyl terminal region around V3. There were more amino acid substitutions overall in the SVR and BT patients than were observed in the NR patients. Six NR patients, listed as 7–12, demonstrated no mutations whatsoever in this region.