首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
HCV infection in more than 200 million individuals worldwide is a principal health problem. Prior to the development of HCV protease inhibitor combination therapy, HCV infected patients were treated with pegylated interferon-α and ribavirin. The adverse side effects associated with this type of treatment may lead to the discontinuation of treatment in certain number of patients. Currently, the inhibitors of NS3/4A Protease were found promising candidates for the treatment of HCV infection. There are several inhibitors of HCV NS3/4A protease that are passing through clinical improvement showing good potency against HCV infections in a number of patients. To further recognize binding interactions and activity trend, the molecular docking studies were performed on a number of HCV NS3/4A protease ketoamide inhibitors via MOE docking protocol. The docking analysis resulted in the detection of important ligand interactions with respect to binding site of target proteinand produced good correlation coefficient (r2 = 0.690) between docking score and biological activities. These molecular docking results should, in our view, contribute for further optimization of ketoamide derivatives as NS3/4A protease inhibitors.  相似文献   

2.
Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.  相似文献   

3.
W Xue  M Wang  X Jin  H Liu  X Yao 《Molecular bioSystems》2012,8(10):2753-2765
Hepatitis C virus (HCV) bifunctional NS3/4A is an attractive anti-HCV drug target, as both the protease and helicase functions are required for viral infection and replication. Although the first generation of NS3/4A protease inhibitors (PIs) has focused almost exclusively on the interaction with the protease domain alone, recent studies have shown that PIs also inhibit the full-length NS3/4A protein. However, the detailed molecular mechanism of the interaction between protease inhibitors, as well as the peptide substance with the full-length NS3/4A protein, remains poorly understood. Herein, starting from the recently determined crystal structure of an inhibitor (inhibitor ) bound to the full-length NS3/4A protein, the structures of the full-length NS3/4A complexed with inhibitor ITMN-191 (by InterMune/Roche; Phase II) and substrate 4B5A (the viral cleavage product peptide) were built. Then, residue interaction network (RIN) analysis, molecular dynamics (MD) simulation, binding free energy calculation, decomposition of free energies on per-residue and dynamic substrate recognition pattern analysis were employed to uncover the structural and energetic basis of inhibitor and substrate binding mode in the binding cleft located at the interface of the protease and helicase domains of the full-length NS3/4A. The results from our study reveal that both the protease and helicase residues of the NS3/4A participate in the interactions with the inhibitor , ITMN-191 and 4B5A. Additional analysis of the NS3/4A substrate and inhibitor envelopes reveals the areas where the consensus inhibitor volume extended beyond the substrate envelope. These areas correspond to drug resistance mutations including Arg155, Ala156 and Asp168 at the protease active site as well as the two conserved helicase residues Gln526 and His528 that strongly interact with the inhibitors. Thus, the findings of this study will be very useful for understanding the interaction mechanism between the inhibitor (substrate) and NS3/4A and also for the rational design and development of new potent molecules targeting the full-length NS3/4A.  相似文献   

4.
Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.  相似文献   

5.
The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.  相似文献   

6.
The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC50]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC50) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.  相似文献   

7.
Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.  相似文献   

8.
Direct acting antivirals have dramatically increased the efficacy and tolerability of hepatitis C treatment, but drug resistance has emerged with some of these inhibitors, including nonstructural protein 3/4?A protease inhibitors (PIs). Although many co-crystal structures of PIs with the NS3/4A protease have been reported, a systematic review of these crystal structures in the context of the rapidly emerging drug resistance especially for early PIs has not been performed. To provide a framework for designing better inhibitors with higher barriers to resistance, we performed a quantitative structural analysis using co-crystal structures and models of HCV NS3/4A protease in complex with natural substrates and inhibitors. By comparing substrate structural motifs and active site interactions with inhibitor recognition, we observed that the selection of drug resistance mutations correlates with how inhibitors deviate from viral substrates in molecular recognition. Based on this observation, we conclude that guiding the design process with native substrate recognition features is likely to lead to more robust small molecule inhibitors with decreased susceptibility to resistance.  相似文献   

9.
K E Reed  A Grakoui    C M Rice 《Journal of virology》1995,69(7):4127-4136
Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.  相似文献   

10.
Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.  相似文献   

11.
Telaprevir, a novel hepatitis C virus (HCV) NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients infected with HCV. However, drug-resistant HCV variants were detected in vivo at relatively high frequency a few days after drug administration. Here we use a two-strain mathematical model to explain the rapid emergence of drug resistance in HCV patients treated with telaprevir monotherapy. We examine the effects of backward mutation and liver cell proliferation on the preexistence of the mutant virus and the competition between wild-type and drug-resistant virus during therapy. We also extend the two-strain model to a general model with multiple viral strains. Mutations during therapy only have a minor effect on the dynamics of various viral strains, although they are capable of generating low levels of HCV variants that would otherwise be completely suppressed because of fitness disadvantages. Liver cell proliferation may not affect the pretreatment frequency of mutant variants, but is able to influence the quasispecies dynamics during therapy. It is the relative fitness of each mutant strain compared with wild-type that determines which strain(s) will dominate the virus population. This study provides a theoretical framework for exploring the prevalence of preexisting mutant variants and the evolution of drug resistance during treatment with other HCV protease inhibitors or polymerase inhibitors.  相似文献   

12.
The emerging of hepatitis C virus (HCV) resistant strains has been considered as a main drawback of the available drugs. Since HCV has a large inactive surface, we would like to hypothesis that the mutation occur in HCV is minimal and causing less resistance against inhibition. In this study, a short peptide inhibitor of HCV namely plectasin was identified by HCV NS3-4A serine protease assay. Plectasin peptide showed considerable inhibition against HCV NS3-4A serine protease. Enzymatic activity of the recombinant NS3-4Apro was analysed by fluorescence release from several fluorogenic peptide substrates which resembling the dibasic cleavage site sequences of the flavivirus polyprotein precursor. Of all amc-labelled peptides, Pyr-RTKR-amc was the most efficiently cleaved substrate with the lowest Km value of 20 µM. The kinetic assay showed that plectasin peptide inhibited NS3-4Apro activity with an IC50 value of 4.3 μM compared to the aprotinin as a standard proteases inhibitor with an IC50 of 6.1 μM. From the results, plectasin peptide also demonstrated a dose-dependent inhibition of HCV replication with a considerable reduction in RLuc activity at 15 µM using HCV replicon- containing Huh-7 cells. Our study has identified a unique natural peptide that can be used to highlight novel structures for the development of drug derivatives with high efficacy of HCV NS3-4A protease inhibitors.  相似文献   

13.
Chronic hepatitis C virus (HCV) infection is a major global public health problem. HCV infection is supported by viral strategies to evade the innate antiviral response wherein the viral NS3.4A protease complex targets and cleaves the interferon promoter stimulator-1 (IPS-1) adaptor protein to ablate signaling of interferon alpha/beta immune defenses. Here we examined the structural requirements of NS3.4A and the therapeutic potential of NS3.4A inhibitors to control the innate immune response against virus infection. The structural composition of NS3 includes an amino-terminal serine protease domain and a carboxyl-terminal RNA helicase domain. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by retinoic acid-inducible gene-I (RIG-I) or melanoma differentiation antigen 5 and suppressed the downstream activation of interferon regulatory factor-3 (IRF-3) and nuclear factor kappaB (NF-kappaB) through the targeted proteolysis of IPS-1. This regulation was abrogated by truncation of the NS3 protease domain or by point mutations that ablated protease activity. NS3.4A protease control of antiviral immune signaling was due to targeted proteolysis of IPS-1 by the NS3 protease domain and minimal NS4A cofactor. Treatment of HCV-infected cells with an NS3 protease inhibitor prevented IPS-1 proteolysis by the HCV protease and restored RIG-I immune defense signaling during infection. Thus, the NS3.4A protease domain can target IPS-1 for cleavage and is essential for blocking RIG-I signaling to IRF-3 and NF-kappaB, whereas the helicase domain is dispensable for this action. Our results indicate that NS3.4A protease inhibitors have immunomodulatory potential to restore innate immune defenses to HCV infection.  相似文献   

14.
In vitro study of the NS2-3 protease of hepatitis C virus.   总被引:3,自引:1,他引:2       下载免费PDF全文
Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.  相似文献   

15.
We recently reported a new class of inhibitors of the chymotrypsin-like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S' site of the protease, which is not generally used by the natural substrates. The effect of prime-site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis-mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S' site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S' site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N-terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P-P' inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.  相似文献   

16.
A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.  相似文献   

17.

Background

Proteases of human pathogens are becoming increasingly important drug targets, hence it is necessary to understand their substrate specificity and to interpret this knowledge in practically useful ways. New methods are being developed that produce large amounts of cleavage information for individual proteases and some have been applied to extract cleavage rules from data. However, the hitherto proposed methods for extracting rules have been neither easy to understand nor very accurate. To be practically useful, cleavage rules should be accurate, compact, and expressed in an easily understandable way.

Results

A new method is presented for producing cleavage rules for viral proteases with seemingly complex cleavage profiles. The method is based on orthogonal search-based rule extraction (OSRE) combined with spectral clustering. It is demonstrated on substrate data sets for human immunodeficiency virus type 1 (HIV-1) protease and hepatitis C (HCV) NS3/4A protease, showing excellent prediction performance for both HIV-1 cleavage and HCV NS3/4A cleavage, agreeing with observed HCV genotype differences. New cleavage rules (consensus sequences) are suggested for HIV-1 and HCV NS3/4A cleavages. The practical usability of the method is also demonstrated by using it to predict the location of an internal cleavage site in the HCV NS3 protease and to correct the location of a previously reported internal cleavage site in the HCV NS3 protease. The method is fast to converge and yields accurate rules, on par with previous results for HIV-1 protease and better than previous state-of-the-art for HCV NS3/4A protease. Moreover, the rules are fewer and simpler than previously obtained with rule extraction methods.

Conclusion

A rule extraction methodology by searching for multivariate low-order predicates yields results that significantly outperform existing rule bases on out-of-sample data, but are more transparent to expert users. The approach yields rules that are easy to use and useful for interpreting experimental data.  相似文献   

18.
Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.  相似文献   

19.
20.

Background & Aims

Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment.

Methods

Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline.

Results

Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing.

Conclusion

In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号