Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

Abstract

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR 237-276 ) sequence associated with IFN resistance was not found, although the presence of Ala 245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a-and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.Hepatitis C virus (HCV) is a major cause of chronic liver disease leading to cirrhosis worldwide and is now recognized as a leading indication for orthotopic liver transplantation in the United States. The HCV genome has a high degree of genetic variability. Interindividual HCV sequence variability has led to the classification of at least six genotypes, while intraindividual variability is referred to as a quasispecies, which usually consists of a predominant viral variant and a variable mixture of highly genetically related yet distinct variants (23, 36).Interferon (IFN) monotherapy leads to sustained virological responses in less than 20% of chronic hepatitis C cases (47). The recent introduction of IFN plus ribavirin combination therapy has significantly improved response rates (8, 38). In multivariate analyses, HCV genotype and high viral load are two important virologic factors that independently predict the likelihood of treatment failure (37, 39)...

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

“…This sequence has been termed the interferon sensitivity-determining region (ISDR). These results were then confirmed by other Japanese studies [11][12][13][14][15] , but were never in accordance with most Western European [16][17][18][19][20] and American studies [21][22][23] , in which most of the ISDR sequences harbored an intermediate profile.…”

“…HCV genotype 1, particularly genotype 1b, are most resistant to combined IFN-R therapy [5] . Among viral factors, genetic variability has been studied in many regions of the HCV genome, mainly in hypervariable region 1 (HVR1) and PKR-eIF2α phosphorylation homology domain (PePHD) of E2 region and in PKRbd (including ISDR) of NS5A [17,22,[39][40][41][42][43] . However, few reports have studied the complete NS5A region [17,22] .…”

High Amino Acid Variability Within the Ns5a of Hepatitis C Virus (Hcv) Is Associated With Hepatocellular Carcinoma in Patients With Hcv–1B-Related Cirrhosis

“…15,16,[34][35][36][37][38] ‡According to references. [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] found between patients with and without HCC. When compared with the deduced consensus sequence derived from all sequenced isolates, 3 or more aa changes within the PKR-bd were observed in 60% of patients with HCC, but only in 6% of those without evidence of tumor.…”

Early changes in hepatitis C viral quasispecies during interferon therapy predict the therapeutic outcome

“…Among the latter, viral resistance to IFN is considered to play a major role, although the molecular basis of such resistance has not been fully defined (8)(9)(10). A specific region of the nonstructural 5A gene (11) has been suggested as a possible determinant of IFN sensitivity (12,13), but this hypothesis is still highly controversial (14)(15)(16)(17)(18)(19). More recently, a second region of potential interest has been identified within the envelope 2 glycoprotein (E2) (20), but its role in determining IFN resistance also remains unclear (21)(22)(23)(24).…”