Is investigation of hepatitis C virus NS5A gene heterogeneity a tool for predicting long‐lasting response to interferon therapy in patients with HCV‐1b chronic hepatitis?

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) may repress the interferon (IFN)-induced protein kinase R (PKR). High variability of different regions in the carboxy-terminal half of NS5A implicated in the interaction with PKR (particularly the interferon sensitivity determining region (ISDR)) may be a predictor of response to IFN in patients infected with genotype 1b of HCV. We examined pretreatment serum samples from 17 HCV-1b infected patients included in the same schedule of IFN therapy. Seven patients were a rare series of sustained responders (SR) with a post-treatment follow-up of 5-7 years, while ten were nonresponders (NR). The carboxy-terminal half of the NS5A gene was amplified and directly sequenced in all 17 cases. In addition, the entire NS5A gene and the part of the HCV E2 gene coding for the hypervariable region 1 (HVR1) were amplified, cloned and sequenced in six cases (three NR and three SR). No difference in number and distribution of amino acid mutations was observed between isolates from SR and NR in any portion of the protein, including the ISDR region. Analysis of full length NS5A confirmed no difference between the two groups. The NS5A gene sequence was different among the six cases cloned although it appeared to be conserved in each individual patient independently of the quasispecies complexity evaluated through HVR1 examination. These data indicate that pretreatment analysis of theNS5A genomic variability has no value in predicting long-lasting response to IFN therapy in HCV-1b-infected patients, and that the HCV NS5A gene has high quasispecies homology.     

Analysis of sequence configurations of the ISDR,PKR-binding domain,and V3 region as predictors of response to induction interferon-alpha and ribavirin therapy in chronic hepatitis C infection

Interferon (IFN) and ribavirin combination therapy for chronic hepatitis C virus (HCV) infection yields a sustained response rate of only 40%. Previous studies have linked IFN responsiveness to viral sequence variation in parts of the E2 and NS5A genes, but this remains controversial. We studied pretreatment sera from 28 subjects (23 with HCV genotype 1a) who received high-dose IFN induction followed by IFN–ribavirin combination therapy. Serum HCV sequences were amplified and compared from 14 responders with undetectable HCV RNA 24 weeks after therapy and 11 nonresponders (excluding three who dropped out of the study). Analysis included the E2 PKR eIF-2 phosphorylation homology domain (PePHD, codons 659–670), where the sequence was well conserved, and codons 2001–2420 of NS5A. In NS5A, the proposed PKR binding domain (codons 2209–2274), containing the putative IFN sensitivity determining region (ISDR, codons 2209–2248), showed too little variation among subjects to differentiate responders and nonresponders. NS5A codons 2356–2385 (which includes the V3 region) exhibited more variation. Here, six of 12 genotype 1a responders showed four or more amino acid changes from the prototype HCV-1 sequence, as compared with one of eight nonresponders, but this fell short of statistical significance (P = 0.16). NS5A sequences from posttreatment sera were examined in six nonresponders to look for selection of treatment-resistant viral subpopulations, but no consistent change was detected. In conclusion, our results indicate that the sequences of the ISDR, the PKR-binding domain, and the PePHD are unlikely to have predictive value for IFN treatment success in those infected with HCV genotype 1a. However, the finding of greater variability among treatment responders in the carboxy end of NS5A suggests that the V3 region merits further investigation.     

Differential effect of interferon on hepatitis C virus 1b quasispecies in the nonstructural protein 5A gene.

A close correlation was reported between amino acid mutations in the nonstructural protein 5A (interferon [IFN] sensitivity-determining region [ISDR]) of hepatitis C virus (HCV)-1b and the response to IFN therapy. The dynamic change of ISDR quasispecies during IFN treatment was investigated in 22 patients. In 18 nonresponders, the number of ISDR mutations in major quasispecies decreased after therapy (P=.039). In each nonresponder, the percentage of wild-type (no mutations in the ISDR) quasispecies increased after treatment (P=.008), whereas the percentages of intermediate- (1-3 mutations) and mutant-type (>/=4 mutations) quasispecies decreased (P=.037 and P=.043, respectively). No mutant-type quasispecies were detected after therapy. Four complete responders had only quasispecies with >/=3 mutations before therapy. Thus, HCVs have fewer mutations in the ISDR after IFN therapy than those before therapy. These IFN-resistant HCVs were already present before therapy as minor quasispecies and were selected by IFN in nonresponders.     

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

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 clon- ing and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-α2b for 3 mo followed by IFN-α2b alone or com- bined IFN-R therapy for 9 additional months. Patients were categorized intro two groups based on their re- sponse to the treatments: 7 with sustained virological re- sponse (SVR) (quasispecies = 150) and 3 non-respond- ers (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 quasispe- cies during therapy. Analysis of amino acids substitu- tions 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 thetwo groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients. CONCLUSION: These results suggest that detailed mo- lecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.     

Mutations in the NS5A and E2‐PePHD regions of hepatitis C virus genotype 1b and response to combination therapy of interferon plus ribavirin

Background/aims: Combination therapy with interferon (IFN) and ribavirin is the current standard treatment for chronic hepatitis C, but the efficacy is still not satisfactory, especially for genotype 1b. NS5A and E2 proteins of hepatitis C virus (HCV) may repress the IFN‐induced RNA‐dependent protein kinase (PKR), and thus have the potential to influence the response of HCV to IFN therapy; however, this issue remains controversial. Methods: Nucleotide sequences of the PKR‐eIF2α phosphorylation homology domain (E2‐PePHD) and PKR‐binding domain (NS5A‐PKR bd) of the HCV genome were analyzed by amplification and direct sequencing in 30 HCV genotype 1b patients who had been treated with IFN and ribavirin. Results: Nine (30%) patients achieved a sustained virological response (SVR) to combination therapy. Pretreatment variables and amino acid substitutions were compared between responders and non‐responders. The responders were younger than non‐responders (37.2±10.4 vs. 45.4±9.5 years, P=0.017), whereas no significant statistical differences were found in the number of amino acid substitutions in NS5A and E2‐PePHD regions between the two groups. Conclusions: Genetic heterogeneity in NS5A and E2‐PePHD regions of the HCV genome may not serve as a predictor for treatment outcome with combination therapy in Taiwanese patients with chronic HCV genotype 1b infection.     

Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and response to interferon therapy

Hepatitis C virus (HCV) genotype 1b and high pretreatment virus load are well known predictive factors of poor response to interferon (IFN) therapy. In addition, a sparsity of amino acid substitutions in the interferon sensitivity determining region (ISDR) is also predictive of a poor response to IFN in patients with genotype 1b, although this issue is still controversial. Recently, a 12 amino acid domain in the E2 protein of HCV (PKR-eIF2 alpha phosphorylation homology domain [PePHD]) has been reported to bind with and block the virus replication inhibition ability of PKR, suggesting that the interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of IFN. To clarify the significance of amino-acid sequences in this domain in predicting the effect of IFN therapy, we analyzed 82 patients with genotype 1b. Eleven patients (13.4%) responded to treatment whereas the remaining 71 patients (86.6%) were nonresponders. Multivariate analysis showed that only HCV load and amino-acid substitutions in the ISDR were predictive of sustained response to IFN. Amino-acid substitutions in the PePHD were detected in only eight of 82 patients (9.8%), and did not correlate with the therapeutic effect of IFN. However, amino-acid-sequence analyses of quasispecies before and after 1 week of IFN therapy showed elimination of clones with substitutions in this domain. Our results suggest that amino-acid sequences of the PePHD domain may be related to viral resistance to IFN but do not predict the outcome of IFN therapy as amino-acid substitutions in this domain are rare.     

Correlation between mutations in the core and NS5A genes of hepatitis C virus genotypes 1a, 1b, 3a, 3b, 6f and the response to pegylated interferon and ribavirin combination therapy

Several studies have reported correlation between mutations in core and NS5A proteins of hepatitis C virus (HCV) and response to interferon (IFN) therapy. In particular, mutations in NS5A protein have been shown to correlate with responsiveness to IFN treatment of HCV-1b in Japanese patients. This study investigated whether amino acid (aa) mutations in the core and NS5A proteins of HCV-1a, 1b, 3a, 3b and 6f correlated with the response to pegylated interferon (Peg-IFN) plus ribavirin (RBV) therapy in Thai patients. The entire sequences of core and NS5A of HCV from 76 HCV-infected patients were analysed in comparison with corresponding reference sequences. The data revealed that the number of aa mutations in full-length NS5A, its C-terminus, IFN sensitivity-determining region, variable region 3 (V3) and V3 plus flanking region of HCV-1b NS5A protein were significantly higher in responders than in the treatment failure group (P = 0.010, 0.031, 0.046, 0.020 and 0.006, respectively). Similar results were found in a putative protein kinase R binding domain region in HCV-6f NS5A protein (P = 0.022). Moreover, specific aa substitutions in NS5A that appeared to be associated with responders or the treatment failure group were observed at positions 78 and 305 for HCV-1b (P = 0.028), 64 and 52 for HCV-1a (P = 0.033) and 6f (P = 0.045). Nevertheless, analysis of aa sequences of core protein revealed highly conserved sequences among HCV genotypes and no significant differences between the viruses from responders and the treatment failure group. Our findings indicate that mutations in aa residues of NS5A of HCV-1a, 1b and 6f correlated well with responsiveness to Peg-IFN and RBV combination therapy.     

Selection of HCV NS5A quasispecies during IFN therapy in patients with chronic HCV

Interferon treatment of chronic hepatitis C virus infection is successful in a minority of patients. The sequence of the interferon sensitivity determining region (ISDR) of the NS5A protein may determine the outcome of therapy in patients infected with HCV genotype 1. To determine whether IFN treatment caused selection of ISDR quasispecies and whether sequences bearing the putative IFN-resistance motif (HCV-J sequence) were selected, we examined amino acid changes in the ISDR in patients with HCV of different genotypes with and without therapy. We found that the ISDR sequence was highly variable and variability was greatest in patients with HCV of genotype 1. IFN treatment was found to exert a selection pressure on ISDR quasispecies, but the putative interferon-resistant variant was not enriched in patients of any genotype. Hence factors other than the sequence of the ISDR region played a role in the IFN resistance of these patients.     

The nonstructural 5A protein of hepatitis C virus genotype 1b does not contain an interferon sensitivity-determining region

BACKGROUND: The nonstructural (NS) 5A protein of hepatitis C virus (HCV) has been suggested to contain an interferon (IFN) sensitivity-determining region (ISDR). METHODS: We studied whether the degree of viral decline on day 1 is associated with differences in NS5A amino acid sequences among patients receiving IFN- alpha. RESULTS: Phylogenetic analyses of the full-length protein and of functional domains showed no relationship between the baseline protein sequence and the antiviral response. NS5A quasispecies sequences showed no differences in the number of mutations in the putative ISDR relative to a prototype sequence between responders and nonresponders or according to IFN- alpha antiviral efficacy. No relationship was found between antiviral efficacy at 24 h and the baseline sequence of any NS5A region. Amino acid changes were observed in a few cases at 24 h in both responders and nonresponders, but no consistent pattern of amino acid shifts was observed, ruling out the possibility that IFN- alpha selected IFN-resistant variants. CONCLUSION: Our findings show that there is no ISDR in the HCV genotype 1 NS5A protein and that the NS5A sequence does not influence the capacity of IFN- alpha to block viral replication. The findings do not rule out a role for NS5A in subsequent viral clearance.     

Evidence for sequence selection within the non-structural 5A gene of hepatitis C virus type 1b during unsuccessful treatment with interferon-alpha

Resistance of the hepatitis C virus (HCV) to interferon-α (IFN-α) therapy in patients with hepatitis C may be genetically controlled by an IFN sensitivity-determining region (ISDR) within the non-structural 5A (NS5A) gene. To assess whether HCV 1b strains carrying a 'resistant' type of ISDR are selected during unsuccessful IFN therapy, we analysed the evolution of the NS5A quasispecies, as detected by the clonal frequency analysis technique, and of the ISDR sequence by nucleotide sequence determination, in 11 patients showing no virological response during two consecutive cycles of IFN-α therapy. IFN-resistant patients had a homogeneous ISDR quasispecies with sequences identical to those described as 'resistant-' or 'intermediate-' type ISDR. After retreatment with IFN, further selection towards a homogeneous viral population was observed and 10 out of 11 patients had only one variant of HCV with no or just one single amino acid mutation within the ISDR sequence. Treatment and retreatment with IFN was associated in our non-responder patients with evolution of the ISDR quasispecies towards a rather homogeneous viral population carrying a conserved or minimally mutated ISDR motif, supporting the idea that this motif may be relevant for IFN resistance in HCV 1b-infected individuals.     

NS5A mutations predict biochemical but not virological response to interferon-α treatment of sporadic hepatitis C virus infection in European patients

The NS5A region of the hepatitis C virus (HCV) genome has been reported by Japanese but not European investigators to be a significant factor in predicting interferon (IFN) response patients with HCV of genotype 1. We correlated the NS5A region with treatment outcome in patients with sporadic HCV infection. Twenty‐eight patients (10 men, 18 women, mean age 60 ± 2 years) with histologically proven HCV chronic hepatitis, genotype 1b, were treated with 6 MU IFN‐α for 6 months. The 6954–7073 area of the NS5A region was directly sequenced for nucleotide and amino acids mutations and the results were related to biochemical and virological response. None of the patients had a strain with nucleotide sequence identical to the Japanese HCV‐J. However, in five strains the nucleotide mutations led to synonymous amino acids and the amino acid sequences were identical to the prototype Japanese strain. Only 2/28 patients had four or more amino acid mutations (mutant strains) while 21 demonstrated an intermediate type and five belonged to the wild‐type. The most frequent non‐synonymous substitution was at position 6982 (A→G) corresponding to an amino acid change at codon 2218 (His→Arg). All patients with the wild‐type were biochemical nonresponders while the two patients with the mutant strains had a sustained biochemical response. Twenty‐three percent of the intermediate type had a sustained biochemical response. NS5A mutations predict the biochemical but not the virological response of patients. Virological response was poor and unrelated to the type of HCV strain. Biochemical responders had significantly lower amino acid mutations (1.14 ± 0.19) compared with nonresponders (2.57 ± 1.4, P < 0.003) as well as lower aminotransferase values (P < 0.01). Hence, mutational analysis of the NS5A region showed that our patients have a mutational profile similar to the European studies with a wild‐type that is slightly different from the Japanese HCV‐J sequence. The biochemical, but not the virological response to IFN‐α is similar to the Japanese studies, with no response of the patients with wild‐type sequence, a good response in the limited number of patients with mutant strains and 23% response rate in the patients with intermediate type sequences.     

Relationship of hepatitis C genotype 1 NS5A sequence mutations to early phase viral kinetics and interferon effectiveness

The interferon (IFN) sensitivity-determining region (ISDR) of the hepatitis C virus (HCV) NS5A protein is controversially implicated in determining IFN-alpha-sustained viral response for HCV genotype 1-infected patients. Because the NS5A protein interferes with protein kinase antiviral activity, this study attempted to determine whether ISDR amino acid mutation number would correlate better with IFN effectiveness to inhibit virus production and elicit early virus clearance. Early viral kinetic data from 22 genotype 1-infected patients treated with high-dose IFN was compared with ISDR mutation number. IFN effectiveness and first-phase viral log decline correlated directly with ISDR mutation number (P=.02). Mean mutation number was higher among rapid responders (3.7+/-1.0; n=6) than among nonresponders (1.3+/-1.0; n=16) (P=.001). Also, second-phase viral log decline and calculated hepatocyte death rate correlated directly with ISDR mutation number (P=.01). This supports a dual effector role for NS5A, which regulates virus production and immune clearance early in therapy.     

Circulating IL-2 and IL-10 in Chronic Active Hepatitis C with Respect to the Response to IFN Treatment

Summary Background: The importance of circulating immunoregulatory cytokines in response to IFN treatment and the change of in vivo production of these cytokines during interferion (IFN) treatment are not well known. We aimed to determine whether pretreatment serum levels of IL-2 and IL-10 are predictive of the response to IFN treatment and to investigate if treatment response or nonresponse has any effect on the circulating levels of these cytokines. Patients and Methods: 37 patients (18 responders and 19 non-responders) with chronic hepatitis C virus (HCV) infection who received IFN-α2b for 6 months were studied. Responders were defined by complete alanine aminotransferase (ALT) normalization and loss of HCV RNA as detected by bDNA assay while patients who had elevated ALT levels and positive HCV RNA after 6 months were considered as nonresponders. Results: Genotype distribution, ALT and HCG RNA levels were similar in responders and nonresponders. A significant number of patients with chronic hepatitis C (20/37 = 54%) had elevated IL-2 levels while IL-10 levels were not different from controls. No difference in baseline cytokine levels was observed between responders and non-responders. In the posttreatment serum samples some patients lost their detectable IL-2 or IL-10; some patients developed detectable cytokine levels after treatment irrespective of the treatment response. Conclusion: These results suggest that active liver injury in chronic hepatitis C is associated with increased circulating Th1 cytokine IL-2 but not with Th2 cytokine IL-10 and that circulating levels of these cytokines do not predict the response to IFN treatment. There is no constant and regular change in circulating levels of these cytokines under IFN treatment with respect to treatment response. Received: June 6, 2000 · Revision accepted: July 21, 2000     

Integrity of the NS5A (amino acid 2209 to 2248) region in hepatitis C virus 1b patients non‐responsive to interferon therapy

Abstract: Background/Aims: In hepatitis C virus‐1b, it has been suggested that an amino acid stretch (aa 2209–2248) of the carboxy terminal half of the non‐structural 5A (NS5A) region participates in the response to interferon treatment. We tested the hypothesis that absence of mutations in the NS5A (aa 2209–2248) sequence is required for interferon resistance. We also investigated the importance of different HCV‐1b isolates in interferon response in France. Methods: We determined the NS5A sequences of 70 patients with chronic hepatitis C before IFN therapy and then compared them with HCV‐J prototype sequence. The isolates were determined by NS5B sequencing, the “gold standard” method for genotyping and subtyping. Pre‐therapeutic viral load was also measured. Results: No sustained virological response was observed in the patients without amino acid substitutions in the NS5A (aa 2209–2248) sequence, and in the patients with HCV‐J isolates. Viral load was significantly higher in the patients with no amino acid substitutions in the NS5A (aa 2209–2248) sequence. Conclusions: In HCV‐1b infected patients, an HCV‐J strain with no amino acid substitution in the NS5A (aa 2209–2248) region indicates a poor prognosis for response to IFN therapy. The low interferon response rate in HCV‐1b infection in Europe is probably not due to a difference between isolates.     

Sequence analysis of PePHD within HCV E2 region and correlation with resistance of interferon therapy in Japanese patients infected with HCV genotypes 2a and 2b

OBJECTIVE: Hepatitis C virus (HCV) E2 protein was recently reported to have a double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2alpha (PKR-eIF2alpha) phosphorylation homology domain (PePHD); PKR is induced by interferon (IFN). PePHD interacts with PKR and inactivates it. PePHD could be a predictor for IFN response, like the interferon sensitivity determination region (ISDR) of HCV NS5A. Several groups reported that PePHD is conserved, and mutations in this region do not correlate with IFN response. In this study, we further investigated the amino acid variation of PePHD among four major genotypes and its correlation with IFN response. METHODS: We enrolled 74 patients for this study and determined PePHD sequence of HCV derived from sera of patients infected with HCV genotype 1a (1 patient; nonresponder [NR]), 1b (36 patients; 4 complete responders [CR], 32 NR), 2a (29 patients; 17 CR, 12 NR), and 2b (8 patients; 3 CR, 5 NR). We also analyzed mutations in ISDR of HCV genotype 1b in 31 patients. RESULTS: PePHD had several variations among four genotypes investigated. In patients infected with HCV genotype 1b, PePHD sequence was well conserved and seemed to have no correlation with IFN response. Mutations in ISDR were correlated with IFN response. In patients with HCV genotypes 2a and 2b, PePHD had multiple variations, and one particular motif, "RGQQ-" at the N-terminus, showed a close correlation with IFN resistance. All eight patients with HCV containing this motif were IFN nonresponders. CONCLUSIONS: IFN resistance of HCV correlates with its "RGQQ-" motif at the N-terminus of PePHD in HCV genotype 2a and 2b. PePHD of HCV could be a predictor of IFN resistance in patients infected with HCV genotype 2a and 2b.     

Effect of ribavirin on dynamics of hepatitis C viremia in interferon alpha-treated patiens with response or no response

Combination therapy with interferon-alpha (IFN alpha) plus Ribavirin has been shown to improve the response rate in patients with chronic hepatitis C as compared to IFN alpha alone. However, the mode of anti-viral action of Ribavirin is still unknown. To prove, whether Ribavirin has any additional effect on the decline of hepatitis C viremia during the first weeks of treatment patients with and without combination therapy were compared. Kinetic studies were performed in patients who either responded to IFN alpha alone or IFN alpha plus Ribavirin combination as well as in nonresponders to both forms of therapeutic approaches. 64 IFN alpha naive patients with histologically proven chronic hepatitis C were included in the study. Patients were randomized to receive either IFN alpha-2a (Hoffmann-La Roche) 6 MU thrice weekly or IFN alpha 6 MU tiw plus Ribavirin (Meduna) 14 mg/kg/day for 12 weeks. 37 patients (58%) became HCV RNA-negative (= responders; 17 [46%] with IFN alpha alone, and 20 [54%] with combination therapy). 27 patients remained HCV RNA-positive (= non-responders; 13 [48%] with IFN alpha alone, and 14 [52%] with combination therapy). HCV RNA concentrations were measured in all patients at baseline as well as 1, 2, 4, and 12 weeks after the start of treatment (bDNA assay, Chiron). Using nonradioactive single-stranded conformation (SSCP)-analysis of the HCV hypervariable region 1 we investigated further whether initial viral decline is correlated with changes in viral quasispecies distribution. In primary responders, ribavirin did not influence hepatitis C viremia decline which was of biphasic nature. Also in nonresponders HCV RNA levels decreased after one week of treatment irrespectively of the mode of therapy (mean 10.0 +/- 2.3 to 5.5 +/- 1.1) (phase 1). In the following weeks, however, 2 types of HCV dynamics could be observed (phase 2). In patients with combination therapy, a further reduction of viremia level could be observed, whereas viremia levels in patients with IFN alpha alone slightly increased (week 12: 3.0 +/- 0.5 MEq/mL [combination, n = 15] vs. 7.5 +/- 2.9 MEq/mL [IFN alpha-mono, n = 12]). The individual response of these nonresponder patients showed, however, marked differences (range percentage decline after 4 weeks, 0-98%). Changes in the viral population (quasispecies distribution) as cause of these differences could be excluded by SSCP-analysis of PCR products of the HCV hypervariable region 1. Ribavirin in combination with IFN alpha exerts an additional anti-viral/immunmodulatory effect which manifests itself in phase 2 of hepatitis C viremia decline. The biphasic decline of hepatitis C viremia also observed in IFN alpha-nonresponders can not be explained by the selection of primary IFN alpha-resistant viral variants. The individual differences in the dynamic of hepatitis C viremia observed in the so called "nonresponders" imply that the term "nonresponder" should be redefined, considering our observation that a marked viral decline can occur in these patients.     

Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon

BACKGROUND & AIMS: The NS5A and the E2 proteins of hepatitis C virus (HCV)-1b can bind and inhibit in vitro the interferon (IFN)-induced cellular kinase PKR. The role of such interaction in modulating the antiviral effect of IFN is still controversial. We have analyzed the E2 and the NS5A sequences in HCV-1b-infected patients treated with IFN to assess whether and how different combinations of wild-type and mutant proteins correlated with early and long-term virological response. METHODS: In 30 patients, sequences of pretreatment and on-treatment E2-PePHD and NS5A-PKR binding domain (including the putative ISDR) were analyzed in parallel by sequencing cDNA-polymerase chain reaction products and up to 25 independent clones. RESULTS: The E2-PePHD sequence was highly conserved with a homogeneous quasispecies and was identical in 29 of 30 cases with no association with the pattern of response and no evidence of evolution during therapy. Patients with a mutated NS5A-ISDR had a higher rate of early virological response (67%) than cases with wild-type ISDR (17%). This association was lost in long-term responders (33% vs. 17%). CONCLUSIONS: Although the highly conserved E2-PePHD motif might contribute to reduce IFN responsiveness, variations within this region do not seem to play a role in modulating IFN sensitivity. The NS5A-ISDR sequence influenced the early, but not the sustained response, to IFN, suggesting that other factors may be more important for the long-term outcome of therapy.     

Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment

Summary. Interferon- α (IFN) has been shown to accelerate the evolution of hepatitis C virus (HCV) variants (quasispecies) in nonresponder patients. Different sensitivities of HCV variants to IFN are discussed as a possible mechanism. In the present study, quasispecies were investigated in detail by a newly established and validated direct solid-phase sequencing of the hypervariable region 1 (HVR1), during the initial 3 months of IFN therapy. According to single strand conformation polymorphism (SSCP) analysis, 14 of 26 (54%) virologic nonresponders with quasispecies evolution were identified. Six representative patients with SSCP changes were selected for frequent HVR1 sequencing. Pre-existing variants were identified by cloning and sequencing of the pretreatment serum HCV sample. In one patient the major type was substituted by a minor variant within 3 days of treatment while in the majority of patients the pretreatment major type did not decline before days 26–57 of treatment. Total serum HCV RNA levels remained constant in all patients. In conclusion, although quasispecies evolution during IFN therapy is common, it occurs after a wide range of time intervals after initiation of therapy. Thus, nonresponse to IFN cannot exclusively be explained by changes in the quasispecies.