Interferon Sensitivity Determining Region Sequences Research Articles

Association of interleukin 28B polymorphism and mutations in the NS5A region of hepatitis C virus genotype 2 with interferon responsiveness.

The single nucleotide polymorphism (SNP) of interleukin 28B (IL28B) and the mutations in the NS5A region of hepatitis C virus (HCV) genotype 1 have been associated with response to interferon (IFN) therapy. However, these relationships in patients with HCV genotype 2 are not well understood. The aim of this study was to investigate whether the SNP of IL28B (rs8099917) and amino acid substitutions in the NS5A region in patients with HCV genotype 2 affect the response to IFN and ribavirin combination therapy. The study enrolled 286 patients with chronic hepatitis C genotype 2. Patients received pegylated-IFN-alpha 2b once each week plus oral ribavirin daily for 24 weeks. Of the 286 patients, 215 (75.2%) achieved sustained virologic response (SVR). Rate of SVR was similar in patients with IL28B TT allele (76%) and those with TG or GG alleles (72%). Patients with SVR were younger than those without SVR (P < 0.001). SVR was achieved in 65.9% of patients with wild-type IFN sensitivity-determining region (ISDR) and 83.5% of patients with mutant type (P < 0.001). There were no significant differences in other factors, including sex, alanine aminotransferase, platelet count, HCV viral load, HCV genotype, and IL28B genotype. The factors related to SVR on multivariate analysis were age (P = 0.019) and ISDR (P = 0.003). ISDR sequence variations are significantly associated with IFN responsiveness in patients with HCV genotype 2. The SNP of IL28B was not associated with SVR in patients with HCV genotype 2.

Mutations in the interferon sensitivity determining region and virological response to combination therapy with pegylated-interferon alpha 2b plus ribavirin in patients with chronic hepatitis C-1b infection

Pegylated-interferon-alpha 2b (PEG-IFN) plus ribavirin (RBV) therapy is currently the de-facto standard treatment for hepatitis C virus (HCV) infection. The aims of this study were to analyze the clinical and virological factors associated with a higher rate of response in patients with HCV genotype 1b infection treated with combination therapy. We analyzed, retrospectively, 239 patients with chronic hepatitis C-1b infection who received 48 weeks of combination therapy. We assessed clinical and laboratory parameters, including age, gender, pretreatment hemoglobin, platelet counts, HCV RNA titer, liver histology, the number of interferon sensitivity determining region (ISDR) mutations and substitutions of the core amino acids 70 and 91. Drug adherence was monitored in each patient. We carried out univariate and multivariate statistical analyses of these parameters and clinical responses. On an intention-to-treat (ITT) analysis, 98 of the 239 patients (41%) had sustained virological responses (SVRs). Patients with more than two mutations in the ISDR had significantly higher SVR rates (P<0.01). Univariate analyses showed that stage of fibrosis, hemoglobin, platelet counts, ISDR mutations, serum HCV RNA level, and adherence to PEG-IFN plus RBV were significantly correlated with SVR rates. Multivariate analysis in subjects with good drug adherence extracted the number of ISDR mutations (two or more: odds ratio [OR] 5.181). The number of mutations in the ISDR sequence of HCV-1b (>or=2) is the most effective parameter predicting a favorable clinical outcome of 48-week PEG-IFN plus RBV therapy in patients with HCV genotype 1b infection.

Mutations in the interferon sensitivity‐determining region of hepatitis C virus genotype 2a correlate with response to pegylated‐interferon‐alpha 2a monotherapy

The interferon sensitivity-determining region (ISDR) is thought to be inhibited by the double-stranded RNA-dependent protein kinase (PKR). Several studies have reported a relationship between the ISDR and interferon (IFN) responsiveness. However, this relationship is controversial. The aim of this study was to investigate whether genomic heterogeneity of the ISDR among patients with hepatitis C virus (HCV) genotype 2a affects the response to pegylated-IFN-alpha 2a monotherapy. Eighty patients (47 men, 33 women; mean age: 54.2 +/- 12.9 years) infected with HCV genotype 2a were evaluated. HCV viral loads were determined by real-time PCR. The ISDR (amino acids 2193-2228) was examined by direct sequencing. Thirty-one patients received subcutaneous injections of pegylated-IFN-alpha 2a (180 microg) once weekly for 24 weeks, and 35 patients received injections for 48 weeks. Fourteen patients withdrew from treatment. Of the remaining 66 patients, 51 (77.3%) showed a sustained virologic response. Factors related to sustained virologic response on multivariate analysis were rapid virologic response (negative HCV at 4 weeks; odds ratio: 0.033; 95% confidence interval (95% CI) 0.003-0.363; P = 0.0052) and the number of mutations in the ISDR (odds ratio: 0.025; 95% CI 0.001-0.476; P = 0.0141). There were no significant differences in other factors, including sex, age, aspartate aminotransferase, alanine aminotransferase, platelet count, duration of treatment, and HCV viral load. Rapid virologic response and the ISDR sequence variations are significantly associated with response to pegylated-IFN-alpha 2a monotherapy in Japanese patients with HCV genotype 2a.

Hepatitis C virus and the controversial role of the interferon sensitivity determining region in the response to interferon treatment

The degree of variability of the interferon sensitivity determining region (ISDR) in the hepatitis C virus (HCV) genome has been postulated to predict the response to interferon therapy, mainly in patients infected with subtype 1b, although this prediction has been the subject of a long controversy. This prediction has been tested by analyzing a cohort of 67 Spanish patients infected with HCV genotype 1, 23 of which were infected with subtype 1a and 44 with subtype 1b. A sample previous to therapy with alpha-interferon plus ribavirin was obtained and several clones (between 25 and 96) including the ISDR were sequenced from each patient. A significant correlation between mutations at the ISDR and response to treatment for subtype 1b patients, but not for those infected with subtype 1a, has been detected. Although the results suggest that the same relationship holds true for subtype 1a, lack of statistical power because of the small sample size of this subtype prevented firmer conclusions. However, identical ISDR sequences were found in responder and non-responder patients, suggesting that the stability of the ISDR sequence can occasionally help HCV to evade interferon therapy, although this is not a sufficient condition. More complex interactions, including the ISDR or not, are likely to exist and govern the HCV response to interferon treatment.

Site‐specific mutation of the interferon sensitivity‐determining region (ISDR) modulates hepatitis C virus replication

The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Does sequencing the PKRBD of hepatitis C virus NS5A predict therapeutic response to combination therapy in an Australian population?

Abstract Background and Aim: The presence of four or more amino acid substitutions within the interferon sensitivity determining region (ISDR) of the hepatitis C virus (HCV) genotype 1b NS5A gene determines sensitivity to interferon (IFN) monotherapy in Japanese patients. Resistance of HCV genotype 1 to IFN-alpha has been attributed to the functional inhibition of a RNA dependent protein kinase (PKR) by the HCV NS5A PKR binding domain (PKRBD), which includes the ISDR. The ability of the ISDR and PKRBD sequence to predict a response to IFN-alpha and ribavirin combination therapy was investigated in an Australian population. The sequence of the PKRBD of NS5A, including the ISDR, for the dominant quasi-species of HCV was determined in 37 genotype 1 (genotype 1a: n = 26, genotype 1b: n = 11) and 13 genotype 3a infected patients. The number of PKRBD amino acid substitutions in HCV genotype 1 infected patients with a sustained virological response was significantly higher than that in patients with a non-response to treatment (P = 0.047). It was found that only 2/37 HCV genotype 1 infected patients had four or more amino acid substitutions relative to the prototype ISDR sequence (HCV-J). Importantly, a sustained virological response was not found in any of the HCV infected patients who had a prototype ISDR genotype 1 sequence (n = 5). There are relatively few amino acid mutations within the ISDR of this Western Australian patient population. Patients infected with a HCV genotype 1 prototype sequence should be counseled before receiving combination IFN-alpha and ribavirin therapy as they have a poor response to treatment.

Sequence elements correlating with circulating viral load in genotype 1b hepatitis C virus infection☆

The correlation between hepatitis C virus (HCV) genomic sequences and circulating HCV RNA levels was assessed to investigate the genetic elements affecting viral load. The interferon sensitivity-determining region (ISDR) sequence and the serum viral load were strongly correlated in 226 patients examined. Analysis of the entire HCV genome from six patients (three with a high and the others with a low viral load) with similar ISDR sequences identified several candidate residues associated with viral load. The amino acid (aa) sequences of these candidate residues and flanking regions in 67 additional patients revealed that only the residue at aa 962 varied significantly between the HCV patients with low and high serum loads ( P = 0.042). At this position, alanine was observed more frequently in the patients with a high viral load. In conclusion, our results strongly suggest that serum HCV RNA loads are inversely correlated with amino acid substitutions in the ISDR, and aa 962 was identified as a possible second determinant of serum HCV RNA load.

ISDR Pattern and Evolution in Patients with Chronic Hepatitis C Treated with Standard Or Peg-Ifn plus Ribavirin

The aim of the study was to characterize the interferon sensitivity determining region (ISDR) mutation pattern and its changes at 4 weeks of treatment in a population of patients infected with hepatitis C virus (HCV) genotype 1b receiving standard or PEG-IFN plus ribavirin (RBV), to find possible early correlates of therapy outcome. Forty-five patients with chronic hepatitis due to HCV 1b were treated by PEG-IFN-alpha2b (n=23) or IFN-alpha2b (n=22) plus RBV 1000-1200 mg/day. They were classified 24 weeks after stopping therapy as sustained responders (SR), relapsers (REL) or non-responders (NR). Sixteen patients were SR, 12 REL and 17 NR. ISDR mutations were evaluated by direct sequencing at baseline in all and after 4 weeks in patients with detectable viraemia (n=30). The frequency of the three ISDR types was 26.7% wild-type, 64.4% intermediate-type and 8.9% mutant-type, without significant difference in their frequency in SR, REL and NR, independent of IFN formulation. Average numbers of mutations in SR, REL and NR were 1.88 +/- 0.54, 1.33 +/- 0.33 and 0.94 +/- 0.25, respectively, P>0.05. The baseline number of ISDR mutations was not related to the extent of viral load decline in the first month of therapy. Sequence analysis of ISDR region performed 4 weeks after starting therapy revealed qualitative or quantitative changes of ISDR sequence in only seven patients, without correlation with response. Thus, in our patients the baseline pattern of ISDR was unrelated to treatment outcome. Selection towards a dominant IFN-resistant strain did not occur under standard or PEG-IFN plus RBV.

Correlation between mutations in the interferon sensitivity-determining region of NS5A protein and viral load of hepatitis C virus subtypes 1b, 1c, and 2a.

In the present study, we analyzed the possible relationship between interferon (IFN) sensitivity-determining region (ISDR) sequence variation of various hepatitis C virus (HCV) subtypes and serum HCV titers in Indonesian patients without IFN treatment. The viremia titers (mean +/- standard deviation) of HCV subtype 1b (HCV-1b) isolates with low (three or fewer) and high (four or more) numbers of ISDR mutations were 5.4 +/- 0.6 and 4.2 +/- 0.9 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Similarly, the viremia titers of HCV-1c isolates with low and high numbers of ISDR mutations were 5.3 +/- 0.6 and <3.0 +/- 0.0 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Also, the virus titers of HCV-2a isolates with low and high numbers of ISDR mutations were 4.3 +/- 0.7 and 3.5 +/- 0.4 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Thus, our results demonstrated that virus load in Indonesian patients infected with HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of mutations in the ISDR sequence, implying the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia.

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.

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.

Mutations in interferon sensitivity- determining region of hepatitis C virus: its relation to change in viral load

OBJECTIVE: Interferon sensitivity-determining region (ISDR) in nonstructural region 5A of hepatitis C virus (HCV) genotype-1b has been reported to be associated with viral load. Viral load is usually small in the patients with mutant type (four or more amino acid substitutions, compared with HCV-J) and large in those with wild (identical to HCV-J) or intermediate type (from one to three amino acid substitutions). A possible correlation was investigated between mutations in ISDR and alterations of viral load during the course of disease. METHODS: The sequences of ISDR were determined in eight patients with significant changes of viral load and in 11 patients without changes. RESULTS: In two of the eight patients with significant alterations of viral load, ISDR sequences changed significantly. In one patient whose viral load increased after a course of interferon therapy, the number of substitutions, compared with HCV-J, decreased from five to zero or one; the type of ISDR converted from mutant type to wild or intermediate type. In one patient whose viral load decreased significantly after two courses of interferon therapy, the number of substitutions increased from one to six; ISDR changed from intermediate type to mutant type. In the remaining six patients with changes of viral load and in the other 11 patients without changes, the sequences of ISDR did not change significantly. CONCLUSIONS: The mutations in ISDR are one of the viral factors involved in the changes in viral load during the course of disease, although the majority of other factors involved are still unknown.

Mutations in interferon sensitivity-determining region of hepatitis C virus: its relation to change in viral load.

Interferon sensitivity-determining region (ISDR) in nonstructural region 5A of hepatitis C virus (HCV) genotype-1b has been reported to be associated with viral load. Viral load is usually small in the patients with mutant type (four or more amino acid substitutions, compared with HCV-J) and large in those with wild (identical to HCV-J) or intermediate type (from one to three amino acid substitutions). A possible correlation was investigated between mutations in ISDR and alterations of viral load during the course of disease. The sequences of ISDR were determined in eight patients with significant changes of viral load and in 11 patients without changes. In two of the eight patients with significant alterations of viral load, ISDR sequences changed significantly. In one patient whose viral load increased after a course of interferon therapy, the number of substitutions, compared with HCV-J, decreased from five to zero or one; the type of ISDR converted from mutant type to wild or intermediate type. In one patient whose viral load decreased significantly after two courses of interferon therapy, the number of substitutions increased from one to six; ISDR changed from intermediate type to mutant type. In the remaining six patients with changes of viral load and in the other 11 patients without changes, the sequences of ISDR did not change significantly. The mutations in ISDR are one of the viral factors involved in the changes in viral load during the course of disease, although the majority of other factors involved are still unknown.

Statistical analysis of combined substitutions in nonstructural 5A region of hepatitis C virus and interferon response

Interferon (IFN) therapy (+/- ribavirin) is the only currently available treatment for chronic hepatitis C virus (HCV), but is not effective for a majority of patients. Several studies have correlated IFN response with substitutions in a small region of the nonstructural 5A (NS5A) gene product of HCV, termed the interferon sensitivity determining region (ISDR). Many other studies, however, have been unable to verify this correlation. To address this issue, available data from published studies was used to create a database of 675 individual ISDR sequences. The database was used to analyze substitutions in the ISDR with regard to IFN response. Combined data was analyzed by the chi-square independence test and by logistic regression analysis. Each statistical analysis demonstrated a strong correlation between IFN response and substitutions in the ISDR. A statistically significant correlation was also found between IFN-response and substitutions in ISDRs of combined studies that independently were unable to detect a correlation. The failure of these individual studies to verify a correlation seems to be at least partially due to inadequate sample size. A new model for chi-square analysis is proposed that could allow a correlation between IFN-response and ISDR sequence to be detected for data sets with less statistical power than that required for the current model. The ISDR database was also used to analyze individual substitutions in the ISDR. The results show that IFN-sensitive viruses contain a larger number and more diverse collection of substitutions than IFN-resistant viruses. Substitutions that are most likely to be tolerated or detrimental to NS5A function in the IFN-response mechanism were identified as a first step toward site directed mutagenesis of the ISDR.

Ratio of serum ?-GT/ALT rather than ISDR variability is predictive for initial virological response to IFN-? in chronic HCV infection

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-alpha. Because the proportion of patients responding to therapy with sustained or even just with transient elimination of viral RNA is low, several potential prognostic parameters have been evaluated to predict the outcome of the therapy. The present study aimed to prove the validity of a predictive parameter described previously for initial virological response, namely the ratio of serum gamma-glutamyltransferase/alanine transaminase (gamma-GT/ALT) activity in connection with virus genotypes 1a, 1b, and 3a, prospectively and to compare the predictive value of these combined parameters with amino acid variability within the interferon sensitivity determining region (ISDR). The prospective analysis confirmed previous data on the predictive value of the serum gamma-GT/ALT ratio. Concerning ISDR variability, the majority of ISDR sequences obtained from the mostly nonresponding type 1b-infected individuals (23/28) resembled nonmutant types (27/ 28). Isolates from type 3a-infected patients responding to therapy in the majority of cases (13/ 20) exclusively resembled nonmutant types when compared with databank type 3a sequences, but were mutant when compared with the prototype sequence HCV-J. However, the initial virological responsiveness among both type 1b- and type 3a-infected patients did not correlate to ISDR variability. In contrast, virological responsiveness was closely related to serum gamma-GT/ALT ratio. The data are not necessarily contrary to the concept that the number of amino acid exchanges within the ISDR compared with the prototype HCV-J sequence is related to some extent to IFN-alpha sensitivity. The ratio of serum gamma-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter.

Long-term response to interferon alpha is unrelated to“interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-1b infection

Background/Aims: Contradictory data have been reported about the predictive value of the variability in interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV) genotype-1b on response to interferon-alpha (IFN-α) therapy. The aim of this study was to examine this issue in a series of patients with long-term response to IFN treatment. Methods: We retrospectively analyzed 24 patients with chronic HCV genotype-1b infection treated with IFN-α (total dose median 677, range 216–1350 MU) selected in 6 Italian Liver Units. These patients were defined as true long-term responders (LTR) since they showed persisting biochemical and virological responses to IFN treatment (mean follow-up 38 months). HCV genomes from pretreatment serum samples were amplified and directly sequenced. The ISDR amino-acid sequences obtained were aligned and compared with the published sequence of HCV-J. Results: Amino-acid substitutions were found in 23 of the 24 patients, and 22 of them showed an H to R amino-acid change at codon 2218. Fourteen patients showed only one mutation (at codon 2218), two had 2, five had 3, one had 4 and one had 5 mutations. When we compared the ISDR sequences from the 24 LTR with those of non-responders (NR), we found no significant correlation between the number of mutations and the response to therapy. Conclusions: Our results demonstrate that the persisting efficacy of IFN treatment in patients with chronic HCV is not related to the number of ISDR amino acid susbstitutions of the infecting viruses. Further studies are needed to verify whether other NS5A sequences outside the ISDR might be involved in the mechanisms of IFN resistance.

Mutations in the interferon-sensitivity determining region of hepatitis C virus and transcriptional activity of the nonstructural region 5A protein

Amino acid (aa) mutations in the interferon-sensitivity determining region (ISDR) (aa position 237-276 of the nonstructural region 5A [NS5A] protein consisting of 447 amino acids) of hepatitis C virus (HCV) are related to increased interferon sensitivity and low viral load, but its mechanism has not been clarified. Recently, the NS5A protein has been reported to have a transcriptional activation function, like other viral transactivator proteins known to repress interferon-induced gene expression, and the ISDR overlaps one of the acidic amino acid regions, putative domains conferring this activity. In the present study, we investigated the transcriptional activation function of the ISDR itself and the effect of amino acid mutations in the ISDR on this activity. The full-length or truncated NS5A cDNA with different ISDR sequences was cloned into a yeast or mammalian expression vector to form a fusion protein consisting of the GAL4 DNA-binding domain (GAL4-DBD) and NS5A protein. Following transfection, the transcriptional activities of these constructs were determined using beta-galactosidase (yeast) or chloramphenicol acetyltransferase (CAT) (mammalian cell) reporter gene expression under the control of GAL4 binding sites. In yeast, both the full-length sequence of NS5A-R (a clone with one aa mutation in the ISDR) and NS5A-S (a derivative of NS5A-R with six aa mutations in the ISDR) had no distinguishable transcriptional activity, whereas an amino-terminal deletion construct of NS5A-R (aa position 228-447) lacking 227 aa, showed remarkable activity with the relative value of 117.0 over that of the backbone vector. The same deletion mutant of NS5A-S produced five times higher activity with the relative value of 575.0, indicating that aa mutations in the ISDR profoundly affect this transcriptional activity. In a hepatoma cell line, HuH-7, the transcriptional activity was more prominent with a construct consisting of only the ISDR and short flanking sequences (aa 228-284) than larger deletion constructs of NS5A-R. Analysis using six different ISDR clones revealed that different mutations enhanced this activity to various extent compared with the wild-type ISDR. In particular, site-directed mutagenesis targeted to the aa position 252 showed that this aa residue had profound influence on the activity. These results suggest that the ISDR has a transcriptional activity, and it is enhanced by aa mutations that are also related to decreased viral load and increased interferon sensitivity. The possible association between transcriptional activation and interferon sensitivity or viral replication should be studied further.

Variation in the hepatitis C virus NS5a region in relation to hypervariable region 1 heterogeneity during interferon treatment.

The putative interferon sensitivity determining region (ISDR) in the NS5a region of the hepatitis C virus (HCV) was analyzed in 13 interferon alpha (IFN-alpha) treated patients representing genotypes 1a, 1b, and 2b. These patients had previously been followed longitudinally during treatment with respect to viral load and to virus heterogeneity using the hypervariable region 1 (HVR1) sequence as a marker. In the present study, the NS5a region was analyzed for nonresponders and sustained responders using direct DNA sequencing. While the previous results of analyzing viral composition and load showed evidence of selection, no corresponding selection of specific NS5a ISDR sequences was observed in the nonresponders, and identical ISDR sequences were observed among both sustained responders and nonresponders. Thus, we cannot verify a correlation between ISDR sequence and the observed selection of IFN-alpha-resistant quasispecies demonstrated as a restriction of HVR1 heterogeneity. This indicates that the potential for using ISDR as a diagnostic or prognostic marker during IFN-alpha treatment is limited.

Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity.

Japanese studies have defined the discrete 2209-2248 amino acid region of the non-structural 5A protein (NS5A(2209-2248)) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy-terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.

Evolution of hepatitis C virus quasispecies in hypervariable region 1 and the putative interferon sensitivity-determining region during interferon therapy and natural infection.

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.