Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy

Abstract

Aim: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA-dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear.

Methods: HCV quasispecies were studied by cloning and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-alpha2b for 3 mo followed by IFN-alpha2b alone or combined IFN-R therapy for 9 additional months. Patients were categorized into two groups based on their response to the treatments: 7 with sustained virological response (SVR) (quasispecies = 150) and 3 non-responders (NR) to IFN-R (quasispecies = 106).

Results: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispecies during therapy. Analysis of amino acids substitutions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate the two groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients.

Conclusion: These results suggest that detailed molecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.

Figure 1

Schematic localisation of the six studied regions in NS5A protein. Amino acids positions are numbered according to the HCV-J polyprotein and the NS5A region is within positions 1973-2419.

Figure 2

Phylogenetic tree of NS5A region (codon 1973-2419) of amino acids HCV variants obtained from ten patients at different time of treatment. D0: beginning of the treatment; M3: 3 mo of treatment; SVR: Sustained Virological Responders to IFN-ribavirin therapy; NR: Non-Responders to IFN-ribavirin therapy. HCV-H strain was used as outgroup. The internal node numbers represent the bootstrap values. Distance scale represents 2% of difference between sequences. When all NS5A sequences within a patient at one time point of treatment are grouped in a cluster, this cluster is presented by only one node.

Figure 3

Genetic complexity of HCV quasispecies (A: nucleotide; B: amino acid) calculated by normalized entropy in seven sustained virological responders and three non-responders with median indicated by horizontal bar for each group. P-value calculated by Mann-Whitney U-test.