Impact of Mutations in NS5A of HCV Genotype1b on the Efficacy of the Antivirus Therapy in Patients with Chronic Hepatitis C
|School||Southern Medical University,|
|Keywords||Hepatitis C virus (HCV) Genotype1b Non-structural protein5A(NS5A) Interferon (IFN) Ribavirin (RBV) Replicon|
Background and Objection:Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and the patients frequently progress to cirrhosis and hepatocellular carcinoma (HCC). WHO estimates that approximately180million people, representing3%of the world’s population, are infected with HCV. HCV infection is also a serious public health problem in China, the infection rate was estimated to be3.2%. The total number of Chinese people who had detectable HCV antibodies might exceed40million. The combination therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) is the current standard of care for patients with chronic hepatitis C. Unfortunately, approximately50%of patients infected with hepatitis C virus genotype1b (HCV-lb) are resistant to this treatment regimen.Studies have shown that mutations in several subgenomic regions of non-structural protein5A (NS5A) of hepatitis C virus (HCV) genotype1b, which include the interferon sensitivity-determining region (ISDR, aa2209-2248), the PKR-binding domain (PKRBD, aa2209-2274), the variable region3(V3, aa2356-2379), and the interferon/ribavirin resistance-determining region (IRRDR, aa2334-2379), are associated with response to interferon (IFN) or IFN/RBV therapy. ISDR:In1995, Enomoto et al. first reported a strong correlation between the number of mutations in the so-called IFN sensitivity-determining region (ISDR; amino acids2209-2248) and the efficacy of IFN monotherapy in Japanese patients with HCV genotype lb infection. Their important conclusion is that patients infected with genotype-lb HCV harbouring more than three mutated sites in this region are more likely to become sustained responders than those whose HCV quasispecies harbour few ISDR mutations. There were three groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). This finding was later confirmed by several other Japanese studies. However, the results of studies from Europe and the United States concerning the correlation between ISDR mutations and treatment response are conflicting.PKRBD:In1997and1998, Gale et al. indicated that not only the sequence of ISDR, but also an additional26amino acids after the carboxy terminal of ISDR, is the key for the interaction between NS5A and the RNA-dependent protein kinase (PKR), which is one of the most important host antiviral proteins. This region was termed the PKR-binding domain (PKRBD; amino acids2209-2274). Subsequent studies established a significant association between mutations within the PKRBD of HCV genotype1and a long-term sustained response to IFN and IFN/PEG-IFN plus ribavirin.V3and IRRDR:Recently, Veillo et al, and El-Shamy et al, reported that a number of residue substitutions within the variable region3(V3; amino acids2356-2379), or within the V3region plus its N-terminally flanking region, called interferon/ribavirin resistance determining region (IRRDR; amino acids2334-2379), have also been found to be associated with treatment outcome. In addition, in2009, the mutation number of entire region from ISDR to V3of NS5A (amino acids2209-2379) was believed to influence the treatment response was reported by Bouzgarrou.The classic IFNa signaling pathway is JAK/STAT pathway. By binding to their specific receptors on the surface of target cells, Type I IFNs stimulates a cascade of intracellular signaling pathways that result in the expression of a large number of interferon-stimulated genes (ISGs), Among the products of numerous ISGs, are the IFN-induced, doublestranded (ds) RNA-dependent protein kinase (PKR), the MxA protein, and ISG15et al, of which the antiviral activities have been well characterized. PKR is a double-stranded RNA-dependent serine/threonine protein kinase. Viral dsRNA binds and activates the enzyme through dimerization and phosphorylation. The activated PKR phosphorylates eIF-2a to limit mRNA translation. As a result, viral replication is blocked at the level of protein synthesis. Gale et al. found that the interaction of NS5A and PKR is a potential mechanism of IFN treatment resistance. This interaction requires I SDR plus26additional amino acids at its C-terminal extremity called PKRBD. It results in a disruption of PKR dimerization and, consequently, an inhibition of PKR-mediated eIF-2alpha phosphorylation. So HCV phosphoprotein NS5A binds PKR and confers resistance to the virus against IFNa.Nevertheless, except Japan, the effect of mutations in NS5A on IFN efficacy has not yet been studied well in other nations or regions in Asia, especially in Mainland China. In this study, we examined the effect of amino acid substitutions in NS5A on the outcome of combined interferon/ribavirin therapy in Chinese patients with chronic HCV-lb infection. The analyses focused on ISDR, PKRBD, IRRDR and V3regions of NS5A protein. Meanwile, we chose suitable HCV NS5A fragments to construct the recombinant HCV-lb replicon, and to set up cell culture system with those recombinant HCV-lb replicons. Finlly, we explored the mechanism that the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.Part I. Amplification of the full length NS5A fragment of HCV-1b and construction of pMD18-NS5AMETHODS:A RT-PCR was used to amplify the full-length NS5A genes from clinic blood samples using LA Taq DNA Polymerase. The amplified PCR products were sequenced directly, and the amino acid sequences of PKRBD, ISDR, V3and IRRDR were analysed using MEGA4software with HCV-J as a reference sequence (Access. No. D90208). Meanwile, we chose suitable HCV NS5A fragments to construct the pMD18-NS5A plasmids.The SPSS software (version17.0, SPSS Inc, USA) was used to analyse the data. The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. A P-value<0.05was considered statistically significant. All P values described were two-sided.RESULTS:1. The full length fragments of NS5A of HCV-1b were obtained, cloned and sequenced succefully from CHC patients.2. The mutations rate of ISDR within NS5A of HCV-lb is73.6%in Guangdong area. According to the classification by Enomoto et al, only one patient was infected with the mutant-type strain (≥4mutations)(1.9%),14of53patients (26.4%) were infected with the wild-type ISDR strains (0mutations),38patients (71.7%) were infected with the intermediate-type ISDR strains (1-3mutations). The mutation numbers of PKRBD is5.7±1.4. The mutation numbers of V3is4.9±1.1. The mutation numbers of IRRDR is5.7±1.5.3. The analyses of the mutation of NS5A of HCV-1b and age group, gender and route of transmission showed that:the number of amino acid substitutions in V3and IRRDR were significantly higher in patients≥50years than in those<50years (P=0.041/P=0.033), to reflect that the olders with HCV-lb infection may have more mutation numbers in V3and IRRDR. However, the variability of amino acid sequence in ISDR and PKRBD regions was similar between patients≥50years and those<50years (P=0.222/P=0.081). There were no significant differences between the the number of amino acid substitutions in NS5A and gender and route of transmission (P>0.05).Part II. Impact of mutations in NS5A of HCV genotype lb on the efficacy of the combined PEG-IFN/RBV therapy in Chinese patients with chronic hepatitis C METHODS:A total of53patients with chronic HCV lb infection who received the combination therapy were analyzed retrospectively. All of the patients completed a48-week course of a combination therapy of the pegylated IFN with ribavirin and a24-week follow up. Forty of the53patients were treated with180μg or135μg of pegylated IFN-a-2a subcutaneously once weekly and800-1000mg of ribavirin orally daily. The remaining13patients were treated with80μg of pegylated IFN-a-2b subcutaneously once weekly and800-1000mg of ribavirin orally daily. To analyse the characteristic of impact of mutations in NS5A of HCV genotype lb on the response of the combined therapy, and focus on the analyses of ISDR, PKRBD, IRRDR and V3regions in NS5A protein.The correlations between the mutations of amino acid sequences in NS5A genes of HCV and the clinical outcomes of patients treated with PEG-IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. The Chi square test was used in comparisons of categorical variables. Binary logistic analyses were performed to identify independent factors associated with a SVR. A P-value<0.05was considered statistically significant. All P values described were two-sided.RESULTS:1. Among53patients enrolled in this study,37(69.8%),39(73.6%) and27(50.9%) patients achieved EVR, ETVR and SVR, respectively. We identified that age, PLT, EVR and ETVR were related to SVR (P=0.001,P=0.012, P=0.013and P=0.000). The EVR and ETVR as predictors of the SVR to IFN therapy have been generally acknowledged. PLT as a predictor for SVR could be explained by the difference at the baseline characteristics of patients. Of27patients with SVR,6had cirrhosis (22.2%). In contrast10of the26non-SVR patients had cirrhosis (38.5%). This study suggests that the older patients (>50years old) achieved SVR with lower frequency. But for EVR and ETVR, no significant association between younger patients (<50years old) and those older patients could be observed (P>0.05). This result demonstrated that the younger patients and the older patients could achieve EVR and ETVR with the same frequency. However, during follow-up, the relapse rate of older patients is higher, resulting in a lower SVR at last. Therefore, the older patients with ETVR should be treated with72weeks or more extended therapy to improve SVR.2. The number of amino acid substitutions in ISDR was significantly higher in patients with SVR than in those with non-SVR (1.3±1.1vs.0.6±0.5; P=0.003). patients with≥2mutations in ISDR was more likely to achieve SVR (P=0.005). we demonstrated that the number of mutations in ISDR is closely associated with response to a more effective PEG-IFN/RBV combination therapy in Chinese patients infected with chronic HCV-lb. Interestingly, the mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Comparing with the two Meta-analysises reports, it is very encouraging that we found PEG-IFN/RBV therapy showed substantial improvement on the SVR of CHC patients with intermediate type of HCV ISDR.3. The number of amino acid substitutions in PKRBD was also significantly higher in patients with SVR than in those who did not respond to antiviral therapy (6.1±1.7vs.5.3±0.7; P=0.027). patients with≥6mutations in PKRBD was more likely to achieve SVR (P=0.039). The mutations within the PKRBD of HCV-1are associated with a sustained virological response to PEG-IFN/RBV therapy. The presence of more than six mutations within PKRBD in the pretreatment sera was a useful factor to predict a sustained virological response to combination therapy.4. Despite the high variability in V3, IRRDR and NS5A (ISDR-V3), there was no significant correlation was observed between the number of mutations within these regions and the treatment response from the present study (P=0.819, P=0.949and P=0.244).5. In our study, none of amino acid mutations of NS5A protein was correlated with a sustained virological response (P>0.05). However, frequent amino acid substitutions at positions2218,2257,2259,2260,2262,2265,2268,2270,2271, 2336,2351,2356,2360,2367,2368,2372,2373,2375and2378were observed in Chinese patients.6. Binary logistic analyses were performed to identify predictors of SVR among patients who received PEG-IFN/RBV. It was found that the number of amino acid mutations within ISDR (odds ratio,9.2; P=0.006), and age (odds ratio,0.9; P=0.006) were two independent predictive factors of SVR.Part Ⅲ. Construction of recombinant HCV-1b replicon by replacing NS5A region from patients’serum samplesMETHODS:The on-off plasmid containing sequences of restriction endonucleases of MIu I and Bcl I was designed based on the backbone of robust HCV1b replicon. The full length fragments of HCV NS5A were amplified from different CHC patients, by RT-PCR, cloned into pMD-18vector, and sequenced for analysis of amino acid (aa) mutation of ISDR, PKRBD, V3and IRRDR within NS5A region. If the amplicon from patient without cutting sequences of both MIu I and Bcl I, the NS5A fragment would be re-amplified using primers containing them for double restriction enzyme cutting and inserted into the replicon for replacement. To construct the recombinant HCV-lb replicon by replacing NS5A region from chronic hepatitis C (CHC) patients’ serum samples in Southern China.RESULTS:The on-off plasmid was obtained by cutting with restriction endonucleases of MIu I and Bcl I on the backbone of robust HCV1b replicon. The NS5A fragment would be cutting with double restriction enzyme and inserted into the replicon for replacement. The core region of ISDR-V3of NS5A was replaced into the HCV replicon plasmid and showed right sequencing result. The plug-in typy recombinant HCV replicon for replacement of NS5A region from CHC patients has been successfully constructed, which provides a basis for further research on the biological characteristics of NS5A protein, mechanism of interferon-resistance and antiviral therapy of difficult-to-treat CHC.Part IV. Mechanism of the effect of amino acid substitutions in NS5A on the outcome of IFN-a based antiviral therapyMETHODS:HCV lb recombinant replicon plasmids containing different NS5A fragments were cutted with restriction endonuclease Scal. HCV subgenomic replicon RNA was obtained by transcription and then transfect into Huh7.5.1cell. To detect the transient replication efficiency of recombinant replicons were transfected into Huh7.5.1cells by luciferase assay. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence.The cell colonies which contain HCV RNA were treated with various concentrations of IFNa-2b (0IU/ml,25IU/ml,50IU/ml,100IU/ml,200IU/ml and400IU/ml), RBV (0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml and80μg/ml), and treated with different schemes of IFN/RBV (IFN25IU/ml+RBV10μg/ml, IFN50IU/ml+RBV10μg/ml and IFN100IU/ml+RBV10μg/ml) for48hours. Then, we detected the luciferase activity of cell colonies treated with IFNa-2b, RBV and IFN/RBV. At last, to validate our hypothesis and to explore the possible mechanism.Datas that the cell colonies were treated with various concentrations of IFNa-2b, RBV and IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The variables were summarized as mean±standard deviation (SD). The comparisons of three groups were performed with analysis of variance of factorial design and one-way AN OVA test. Multiple comparisons were performed to identify between-groups differences by LSD or Dunnett’T3methods. A P-value<0.05was considered statistically significant. All P values described were two-sided.RESULTS:1. We detected the transient replication efficiency of recombinant replicons after transfection at4,24,48and72hour sites. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence. These datas certified recombinant replicons could express normally.2. The cell colonies which contain HCV replicon RNA were treated with various concentrations of IFNa-2b and RBV. After that, we found luciferase activity of three groups showed a dose dependent decreasion, accounting that IFN and RBV all could inhibit HCV RNA in replicon cells with a dose dependent enhancement. Interestingly, when cells treated with100IU/ml and200IU/ml of IFNa-2b, luciferase activity of three groups were significant difference (P=0.031and P=0.010). Meanwile, multiple comparisons were performed and showed luciferase activity of NS5A-3group was lowest than those in other groups. This experiment part demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN therapy.3. The cell colonies which contain HCV replicon RNA were treated with three schemes of IFN/RBV. After that, we detected the luciferase activity of three groups, when cells treated with IFN50IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.017). Meanwile, multiple comparisons were performed and showed the luciferase activity of NS5A-1group was highest than those in other groups. It demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN/RBV therapy. when cells treated with IFN100IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.005). Meanwile, multiple comparisons were performed and showed significant differences between every two groups. The luciferase activity of NS5A-3group was lowest than those in other groups, and the luciferase activity of NS5A-2group was higher than that in NS5A-1group. This experiment part demonstrated that HCV was more sensitivity in combined IFN/RBV therapy. And the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy.CONCLUSION: 1. Our results indicated the mutations in NS5A of HCV genotype1b could influence the efficacy of the IFN-a based antivirus therapy in Chinese patients, and the critical regions are ISDR and PKRBD within NS5A protein. We demonstrated that the number of mutations in ISDR was closely associated with response to a more effective PEG-IFN/RBV combination therapy. The mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Our experiment part also demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy than IFN therapy.2. Our results suggested a special attention should be paid to the potential accuracy mechanism of the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.