Genetic diversity of NS5A protein from hepatitis C virus genotype 3a and its relationship to therapy response

Abstract

Background: The quasispecies nature of HCV may have important implications for viral persistence, pathogenicity and resistance to antiviral agents. The variability of one of the viral proteins, NS5A, is believed to be related to the response to IFN therapy, the standard treatment for infection. In this study we analyzed the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a, before IFN therapy.

Methods: Viral RNA was isolated from samples of 12 patients: four sustained virological responders (SVR), four non-responders (NR), and four end-of-treatment responders (ETR). cDNA was synthesized, the NS5A region was amplified and the fragments obtained were cloned. Fifteen clones from each patient were sequenced with eight primers, generating 179 contigs.

Results: Higher values for substitution (either synonymous or non-synonymous) and for distance were found in the SVR group. However, the NR group showed relatively more non-synonymous mutations than the other groups, owing to the higher values of dN/dS in complete NS5A and most specific regions. Overall, NS5A protein is undergoing purifying selection, since all dN/dS ratios values are below 0.5.

Conclusions: Our study provides an overview of the genetic variability of complete NS5A protein in HCV genotype 3a.

Figure 1

Different nucleotide and amino acid sequences. Graphic representation of the different nucleotide (A) and amino acid (B) sequences found in the samples from each patient. The response groups are denoted: SVR -- sustained virological responder, NR -- non-responder, ETR -- end-of-treatment responder.

Figure 2

Mutation sites. Graphic representation of the mutation sites found in each patient, according to the reference sequence for genotype 3, NZL1 (GeneBank D17763). SVR patients are represented in blue, NR patients in pink, and ETR patients in green.

Figure 3

Phylogenetic tree. Unrooted phylogenetic tree constructed with the 179 sequences of full NS5A generated in this study, plus the reference sequence for genotype 3, NZL1 (GeneBank D17763) - Red, the six full-length NS5A sequences from genotype 3a, with country information, available in GenBank (Accession numbers: AY956467; DQ430819; DQ430820; DQ437509; X76918; GQ300882.1) and 20 Brazilian NS5A sequences with 1308 bp (Acession numbers: EF207999.1 to EF208018.1), by the distance method with the Tamura-Nei+I+G model using the neighbor-joining algorithm. Bootstrap was calculated with 1000 replications. SVR -- sustained virological responder - Blue, NR- non-responder - Pink, ETR -- end-of-treatment responder - Green.