Mutations In Interferon Sensitivity-determining Region Research Articles

Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment.

AIMTo determine the number of mutations in the NS5A region of the hepatitis C virus (HCV) and its relationship to the response to antiviral therapy in patients with chronic hepatitis C genotype 1 who are non-responders to two or more treatments.METHODSSequences within HCV NS5A [PKR binding domain (PKRBD) and the interferon-sensitivity-determining region (ISDR)] were analysed via direct sequencing in a selected cohort of 72 patients, with a total of 201 treatments [interferon-alpha (IFN-α), n = 49; IFN-α + ribavirin (RBV), n = 75; pegylated (peg) IFN-α + RBV, n = 47; first-generation direct-acting antivirals (DAAs), n = 13; and second-generation DAAs, n = 17]. Of these, 48/201 achieved a sustained virological response (SVR) and 153/201 achieved no virological response (NVR).RESULTSFor both regions, treatments resulting in SVR were associated with more baseline mutations than were treatments resulting in NVR (SVR vs NVR; PKRBD: 5.82 ± 3 vs 4.86 ± 2 mutations, P = 0.045; ISDR: 2.65 ± 2 vs 1.51 ± 1.7 mutations, P = 0.005). A decrease or no change in the number of mutations over time between treatments in the PKRBD or ISDR, as shown by sequencing, was associated with patients who usually failed to respond to treatment (PKRBD, P = 0.02; ISDR, P = 0.001). Moreover, patients showing a post-treatment baseline viral load > 600000 IU/mL and increased ISDR mutations with respect to the previous treatment were 9.21 times more likely to achieve SVR (P = 0.001).CONCLUSIONThe obtained results show that among patients who have shown no response to two or more antiviral treatments, the likelihood of achieving SVR increases with the genetic variability in the ISDR region (≥ 2 mutations or number of substitutions from the HCV-J and HCV-1 prototype), especially when the viral load is greater than 600000 IU/mL.

Effect of Hepatitis C Virus Genotype 1b Core and NS5A Mutations on Response to Peginterferon Plus Ribavirin Combination Therapy.

We examined whether hepatitis C virus (HCV) genotype 1b core- and NS5A-region mutations are associated with response to peginterferon α-2b plus ribavirin combination therapy. A total of 103 patients with high HCV genotype 1b viral loads (≥100 KIU/mL) were treated with the combination therapy. Pretreatment mutations in the core region and interferon sensitivity determining region (ISDR) in the NS5A region were analyzed. In univariate analysis, arginine and leucine at positions 70 and 91 in the core region, defined as double wild (DW)-type, were associated with early virologic response (p = 0.002), sustained virologic response (SVR) (p = 0.004), and non-response (p = 0.005). Non-threonine at position 110 was associated with SVR (p = 0.004). Multivariate analysis showed the following pretreatment predictors of SVR: hemoglobin level ≥ 14 g/dL (odds ratio (OR) 6.2, p = 0.04); platelet count ≥ 14 × 104/mm3 (OR 5.2, p = 0.04); aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio < 0.9 (OR 6.17, p = 0.009); DW-type (OR 6.8, p = 0.02); non-threonine at position 110 (OR 14.5, p = 0.03); and ≥2 mutations in the ISDR (OR 12.3, p = 0.02). Patients with non-DW-type, non-threonine at position 110, and <2 ISDR mutations showed significantly lower SVR rates than others (11/45 (24.4%) vs. 27/37 (73.0%), respectively; p < 0.001). SVR can be predicted through core and NS5A region mutations and host factors like hemoglobin, platelet count, and AST/ALT ratio in HCV genotype 1b-infected patients treated with peginterferon and ribavirin combination therapy.

Effect of interferon plus ribavirin therapy on hepatitis C virus genotype 3 patients from Pakistan: Treatment response, side effects and future prospective.

More than 10 million people are suffering from hepatitis C virus (HCV) in Pakistan. The available treatment option is a combination of interferon and ribavirin. Treatment response is linked with several factors and also induces a number of side effects. We searched in Pubmed, Pak Medi Net and Google Scholar for the articles presenting the effect of interferon plus ribavirin therapy on HCV patients from Pakistan, their side effects and future prospects. The major prevalent HCV genotype in Pakistan is 3. Conventional interferon alpha plus ribavirin showed sustained virological response of 54%-64% while pegylated interferon alpha plus ribavirin showed sustained virological response of 58%-75%. IL-28B CC genotype is linked with better sustained virological response. Studies on patients with HCV genotype 3 infections showed no correlation between treatment response and interferon sensitivity determining region mutations. Interferon therapy is linked with a number of side effects like thyroid dysfuncton, haematological disorders, weight loss, gastrointestinal tract side effects and neuropsychiatric side effects. Unusual side effects of clubbing of fingers and seizures were also observed in a couple of patients. Interferon alpha plus ribavirin therapy showed better response rate in HCV genotype 3 patients from Pakistan with number of side effects. A couple of interferon free therapies are light of hope for the patients living with HCV.

Prediction of Response to Pegylated Interferon/Ribavirin Combination Therapy for Chronic Hepatitis C Genotypes 2a and 2b and High Viral Load

Objective: We investigated the impact of host genetics represented by the single nucleotide polymorphism (SNP) of the IL28B gene and viral genetic variations within the nonstructural protein 5A (NS5A) [including the interferon (IFN)/ribavirin (RBV) resistance-determining region (IRRDR) and the IFN sensitivity-determining region (ISDR)] on the outcome of pegylated IFN and RBV (PEG-IFN/RBV) treatment. Methods: Sixty-six patients infected with hepatitis C virus (HCV)-2a or HCV-2b who received PEG-IFN/RBV for 24 weeks were examined. Results: In HCV-2a, the major genotype of IL28B SNP showed a tendency toward association with sustained virological response (SVR) and rapid virological response (RVR), and four or more mutations in IRRDR (IRRDR[2a] ≥4) and one or more mutations in ISDR plus its carboxyl-flanking region (ISDR/+C[2a] ≥1) were significantly associated with SVR and RVR. In HCV-2b, one or more mutations in the N-terminal part of IRRDR (IRRDR/N[2b] ≥1) were significantly associated with RVR. Multivariate analysis identified the major genotype of IL28B SNP and IRRDR[2a] ≥4 as independent predictive factors of SVR in HCV-2a, with IRRDR[2a] ≥4 being more powerful than the IL28B SNP. Also, IRRDR[2a] ≥4 in HCV-2a and IRRDR/N[2b] ≥1 in HCV-2b were significant determiners of RVR. Conclusion: The NS5A sequence heterogeneity and IL28B SNP are useful factors to predict the sensitivity to PEG-IFN/RBV therapy in HCV-2a and HCV-2b infections.

Role of hepatitis C virus substitutions and interleukin-28B polymorphism on response to peginterferon plus ribavirin in a prospective study of response-guided therapy

Recent studies have indicated that amino acid (aa) substitutions in the core region and NS5A interferon sensitivity-determining region (ISDR) of hepatitis C virus (HCV) as well as genetic polymorphisms in the interleukin-28B (IL-28B) locus affect the outcome of interferon (IFN)-based therapies. We aimed to investigate the role of these factors on response to peginterferon plus ribavirin in a prospective study of response-guided therapy. The aa sequences in core region and ISDR and rs12979860 genotypes were analysed in 115 HCV-1 patients. The treatment was 24 weeks for patients achieving a rapid virological response (RVR), 48 weeks for those with an early virological response (EVR) and early terminated in those without an EVR. A sustained virological response (SVR) was achieved in 82% of 34 RVR patients, 45% of 74 EVR patients and 0% of seven non-EVR patients. Logistic regression analysis showed that ISDR mutation (≥2) [odds ratio(OR): 6.024], double core 70/91 mutations (OR: 0.136), and platelet counts≥15×10(4) /μL (OR: 3.119) were independent pretreatment factors associated with SVR. Apart from rs12979860 CC genotype, low viral load and ISDR mutation (≥2) were significant factors predictive of RVR. Combination of rs12979860 genotype and baseline viral characteristics (viral load and core/ISDR mutations) could predict RVR and SVR with positive predictive value of 100% and 91%, and negative predictive value of 80% and 54%, respectively. In conclusion, pretreatment screening rs12979860 genotype and aa substitutions in the core region and ISDR could help identifying patients who are good candidates for peginterferon plus ribavirin therapy.

Mutations in different regions of the genome of hepatitis C virus genotype 1b and association with response to interferon therapy

The aim of this study was to investigate the association of mutations in the E2/NS1 [hypervariable regions 1 and 2 (HVR1 and HVR2)] and NS5A regions of the hepatitis C virus (HCV) genome and the effectiveness of interferon (IFN) therapy, and assess whether the degree of heterogeneity of HCV quasispecies predicts response to IFN treatment. Fourteen patients infected with HCV genotype 1b (HCV-1b) who were treated with pegylated IFN-α-2a and ribavirin for 24 weeks, were studied. E2/NS1 and NS5A gene segments were amplified by reverse-transcription polymerase chain reaction. HCV quasispecies heterogeneity in the E2/NS1 region was determined by cloning and sequencing. Mutations in the NS5A region were detected by direct sequencing. The heterogeneity of HCV quasispecies in the HVR1 was significantly greater in the non-responder group than in the responder group, but was not significant for HVR2 or NS5A. The correlation between mutations in IFN sensitivity-determining region (ISDR, NS5A2209-2248) and IFN sensitivity could not be supported. The degree of quasispecies heterogeneity in HVR1, but not in HVR2 and NS5A, may be predictive of response to IFN therapy. An ISDR may not apply to patients infected with HCV-1b.

Mutations in non-structural 5A and rapid viral response to pegylated interferon-α-2b plus ribavirin therapy are associated with therapeutic efficacy in patients with genotype 1b chronic hepatitis C

For patients chronically infected with hepatitis C virus (HCV), mutations in the non-structural 5A (NS5A) gene are important predictive factors for the response to interferon (IFN) therapy. In the present study, factor analysis of the therapeutic response of patients following pegylated IFN and ribavirin combination therapy was assessed in a multicenter study. Chronic HCV-infected patients with genotype 1b and high viral load (n=96, mean age 56.5 years; 59 males, 68 females) treated with pegylated IFN-α-2b and ribavirin combination therapy were enrolled. This study was conducted at Kobe University Hospital and 25 affiliated hospitals in Hyogo prefecture. Sixty-five patients (68%) completed treatment with both pegylated IFN and ribavirin at >80% of the weight-based scheduled dosages. Patients who reduced or terminated therapy were frequently aged women (mean age 60.8 years; 11 males, 17 females). Overall, a sustained viral response (SVR) was achieved in 42 (44%) patients out of 96. Based on per-protocol-based (PPB) analysis, the SVR rate in patients with ≥6 amino acid (aa) mutations in the IFN resistance-determining region (IRRDR) (75%) or ≥1 aa mutation in the IFN sensitivity-determining region (ISDR) (61%) was significantly higher than that in patients with <5 aa mutations in IRRDR (30%) or no mutation in ISDR (29%). Multivariate analysis revealed that rapid viral response (RVR) (odds ratio, 18.1) and mutations of ≥6 in IRRDR (odds ratio, 15.5) were significantly associated with SVR. In conclusion, mutations in the NS5A region, particularly in patients with ≥6 aa mutations in IRRDR were strongly associated with a therapeutic response to pegylated IFN and ribavirin combination therapy.

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 within the interferon sensitivity determining region in Korean patients infected with hepatitis C virus genotype 1b

The treatment response to interferon could differ with mutations in the interferon-sensitivity-determining region (ISDR) in patients infected with hepatitis C virus (HCV) genotype-1b (HCV-Ib). We examined the pattern of ISDR mutations and analyzed whether the number of amino acid substitutions influences the treatment response to peginterferon plus ribavirin in chronic hepatitis or cirrhotic patients infected with HCV-Ib. The study population comprised 52 patients who visited Seoul Asan Medical Center and Seoul National University Bundang Hospital from January 2006 to December 2008 and who received peginterferon alpha-2a (n=37) or -2b (n=15) plus ribavirin, and whose serum was stored. We analyzed the early virologic response, end-of-treatment response, and sustained virologic response (SVR), and examined the ISDR using direct sequencing. The proportions of patients with ISDR mutation types of wild (0 mutations), intermediate (1-3 mutations), and mutant (> or =4 mutations) were 50.0%, 42.3%, and 7.7%, respectively, and the corresponding SVR rates were 63%, 50%, and 67% (p>0.05). The SVR rates were 59.4% and 50.0% in patients with <2 and > or =2 mutations, respectively (p>0.05). On univariate analysis, age was the only predictive factor for SVR (p=0.016). The pretreatment HCV RNA titer tended to be lower in those with SVR, but without statistical significance (p=0.069). The frequency of ISDR mutations was low in our cohort of Korean patients infected with HCV-Ib. Therefore, ISDR mutations might not contribute to the response to treatment with peginterferon plus ribavirin.

Mutations in the interferon sensitivity‐determining region (nonstructural 5A amino acid 2209–2248) in patients with hepatitis C‐1b infection and correlating response to combined therapy of pegylated interferon and ribavirin

Most reports suggest that mutations in the interferon sensitivity-determining region (ISDR) correlate with response to conventional interferon-based therapies in hepatitis C virus-1b (HCV-1b) patients. However, the correlation between ISDR region mutations and response to pegylated interferon plus ribavirin therapy in HCV-1b patients remains unclear. To assess whether ISDR mutations correlate with response to Peg interferon plus ribavirin therapy in HCV-1b patients. Sixty HCV-1b naive patients who had undergone 6 months of Peg interferon alpha-2b plus ribavirin and a 6-month follow-up were enrolled. The amino acid sequences of the nonstructural 5A-interferon-induced RNA-dependent protein kinase (NS5A-PKR)-binding domain were determined by polymerase chain reaction and sequencing. Thirty (50%) patients achieved sustained virological response (SVR). Univariate analysis showed that the proportion of patients with ISDR mutations >or=4 and rapid virological response rate was higher in the sustained virological response group than in the non-SVR group. Viral load was lower in the SVR group than in the non-SVR group. Multivariate analysis revealed that ISDR mutations >or=4 and ribavirin >or=14 mg/kg/day were independent predictors of SVR. Mutations of the ISDR correlate with SVR to Peg interferon alpha-2b plus ribavirin therapy in HCV-1b patients.

Mutations in ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic hepatitis C virus genotype 1b infection

The interferon sensitivity determining region (ISDR) in the non-structural 5A protein (NS5A) gene of hepatitis C virus genotype 1b (HCV-1b) has been demonstrated in Japanese patients; however, contradictory data have been reported from other parts of the world, particularly from Europe and the USA. This study investigated whether mutations in the ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic HCV-1b infection. Forty-five Chinese patients with chronic HCV infection were enrolled in a retrospective study. Of these patients, 20 with HCV-1b infection completed an interferon monotherapy course and 6-month follow-up. The pretreatment mutations in the nucleic acid sequence of NS5A 2209-2248 (ISDR) of HCV-1b were identified by sequencing RT-PCR products followed by comparison with the prototype sequence (HCV-J). Correlation with the clinical efficacy of interferon alpha therapy was then analyzed retrospectively. The statistical analysis indicated a significant correlation between the mutations in ISDR of HCV-1b and sustained virological response to interferon alpha treatment (P < 0.05), and an association of the mutations in ISDR with the pretreatment serum alanine aminotransferase levels (P < 0.01) in Chinese patients with chronic HCV-1b infection. The results support the concept that the response of HCV-1b infected patients towards interferon therapy is influenced by the mutations within the ISDR of NS5A gene.

Mutations in the nonstructural region 5B of hepatitis C virus genotype 1b: Their relation to viral load, response to interferon, and the nonstructural region 5A

The nonstructural 5B (NS5B) protein of hepatitis C virus possesses RNA-dependent RNA polymerase activity and plays an essential role in viral replication. The mutations in NS5B were determined and the correlation with viral load and response to interferon (IFN) were assessed. The entire NS5B region in 33 patients and its thumb domain in 62 patients was sequenced. The number of amino acid substitutions in the NS5B protein, that in thumb domain and the substitution at aa 389 was correlated with viral load and the response to IFN. Multivariate analysis selected only mutation in IFN sensitivity determining region (ISDR) as a factor associated with the viral load and response to IFN. The number of substitutions in the thumb domain and the substitution at aa 389 correlated with the number of substitutions in the ISDR. These results suggest that mutations in NS5B, especially in the thumb domain and at aa 389, have an important effect on viral load and the response to IFN, although they were dependent on mutations in ISDR. Further studies on the relationship between NS5B and NS5A (ISDR) are necessary to elucidate the mechanism of the correlation with viral load and the response to IFN.

Analyse des mutations des domaines ISDR et V3 de la protéine NS5A du virus de l'hépatite C avant le traitement par l'interféron avec ou sans ribavirine

Aim of the study. – The hepatitis C virus (HCV) non-structural NS5A protein has been controversially implicated in the resistance of HCV to interferon therapy in clinical studies. In Japan, mutations in the interferon sensitivity-determining region (ISDR) in the NS5A gene were associated with response to interferon therapy in patients infected with genotype 1b. In contrast, studies from Europe did not confirm such association. More recently, it has been suggested that the V3 domain outside the putative ISDR might also have amino acids changes that may be involved in the resistance to IFN. In this study, the relationship between NS5A mutations in ISDR and V3 domains and virological response to therapy were investigated. Materials and methods. – The NS5A gene was sequenced from 35 HCV genotype 1b infected patients at D0 of a prospective clinical trial of interferon therapy and interferon plus Ribavirin combination therapy. Results. – In the ISDR domain, we did not observe any significant differences in amino acids changes between responders (1.7 ± 1.8, n = 19, range 0–6) and non-responders (1.1 ± 0.8, n = 14, range: 0–3), ( P = 0.483), to therapy before the beginning of treatment. In the V3 domain, we found more mutations in responders (6.5 ± 1.9, range: 2–11) than in non-responders (4.7 ± 1.2, range: 3–8) ( P = 0.0013), before the beginning of treatment. Conclusion. – Our results confirm that, in Europe, the ISDR domain is not predictive for treatment success but suggest that the V3 domain have greater variability in responders than non-responders.

Mutations of the interferon sensitivity-determining region (ISDR) correlate with the complexity of hypervariable region (HVR)-1 in the Japanese variant of hepatitis C virus (HCV) type 1b.

Hepatitis C virus (HCV) genotype 1b comprises mainly two subtypes in Japan, each named for its geographic prevalence (Japan-specific, J type; worldwide, W type). Because the newly identified subtypes have not been fully characterized, the present study directed this issue from virological viewpoints such as hypervariable region (HVR)-1 as well as interferon (IFN) sensitivity-determining region (ISDR). Fifty chronic hepatitis patients with HCV 1b (31 men and 19 women; mean age 50.5 years) were enrolled, and J/W type was determined according to envelope 1 (E1) sequence as described previously (23 J type and 27 W type). Correlations between age, number of HVR-1 clones, HVR-1 diversity, and ISDR mutations were analyzed in J and W type patients independently. In addition, the sequences of the three HCV regions obtained for the determination of the above genetic factors were studied phylogenetically. The number of HVR-1 clones was significantly higher for J type in comparison with W type (P = 0.044). In the J type-infected patients, the ISDR mutation number was correlated inversely with HVR-1 clone number (P = 0.0001, r = -0.734) and HVR-1 diversity (P = 0.0001, r = -0.722). However, this correlation was not observed in the W type patients. W type patients showed a significant correlation between age and HVR-1 clone number (P = 0.015, r = 0.462). Phylogenetic study revealed that the nonstructural (NS) 5A sequence, which is obtained for ISDR type determination, can distinguish between J and W types. The inverse correlation in J type patients between ISDR mutations and HVR-1 complexity may explain the usefulness of the ISDR for prediction of IFN response only in Japanese patients. This suggests that the ISDR is not directly related to IFN responsiveness, but the degree of HVR-1 complexity may be more important.

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.

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.

Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.

The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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.