Subcellular localisation of NS3 in HCV-infected hepatocytes.

Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.     

Allele-specific real-time PCR system for detection of subpopulations of genotype 1a and 1b hepatitis C NS5B Y448H mutant viruses in clinical samples

The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine hepatitis C virus (HCV) polymerase inhibitors in vitro and in vivo. However, the level and the evolution kinetics of this resistance mutation prior to and during treatment are poorly understood. In this study, we developed an allele-specific real-time PCR (AS-PCR) assay capable of detecting Y448H when it was present at a level down to 0.5% within an HCV population of genotype 1a or 1b. No Y448H mutation was detected above the assay cutoff of 0.5% in genotype 1b-infected Con-1 replicons prior to in vitro treatment. However, the proportion of replicons with the Y448H mutation rapidly increased in a dose-dependent manner upon treatment with GS-9190. After 3 days of treatment, 1.2%, 6.8%, and >50% of the replicon population expressed Y448H with the use of GS-9190 at 1, 10, and 20 times its 50% effective concentration, respectively. In addition, plasma from 65 treatment-naïve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively) was tested for the presence of Y448H by AS-PCR and population sequencing. As expected, all patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR results were obtained for 62/65 samples tested, with low levels of Y448H ranging from 0.5% to 3.0% detected in 5/62 (8%) treatment-naïve patient samples. These findings suggest the need for combination therapy with HCV-specific inhibitors to avoid viral rebound of preexisting mutant HCV.     

Hepatitis C virus NS2/3 protease regulates HCV IRES-dependent translation and NS5B RdRp activity

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.     

Internal cleavage of hepatitis C virus NS3 protein is dependent on the activity of NS34A protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) is indispensable for virus replication and a multifunctional enzyme that contains three catalytic activities such as serine protease, helicase, and NTPase. Here, we demonstrated that the internal cleavage of the HCV NS3 protein occurs in various mammalian cells such as HepG2, COS-7, and NIH3T3. As is observed for the internal cleavage mechanism of the NS3 protein of dengue virus 2, the internal processing of HCV NS3 protein was catalyzed by the active NS3 serine protease and NS4A, but not NS3 alone. From the data acquired from extensive site-directed mutagenesis, we observed that the NS3 protein was internally cleaved at two different sites, FCH(1395) ||S(1396)KK and IPT(1428) ||S(1429)GD, within RNA helicase domain. The internal cleavage of NS3 protein by NS34A protease was also confirmed in a different isolate of HCV-1b strain. In addition, in vitro transforming assays demonstrated that the internal cleavage product of NS3, NS3a-1, appeared to have higher oncogenic potential than does intact NS3. Taken together, our results suggest that the internal cleavage of NS3 may be associated with the replication and oncogenesis of HCV.     

Sustained virological response in a patient with chronic hepatitis C treated by monotherapy with the NS3-4A protease inhibitor telaprevir

Here, we describe for the first time a case of sustained virological response (SVR) achieved in a patient with chronic hepatitis C (CH-C) by monotherapy with a NS3-4A protease inhibitor, telaprevir, without interferon therapy. A 59-year-old treatment-naïve Japanese man was enrolled in a phase II trial of telaprevir by repeat oral administration at a dose of 750 mg every 8 h for 24 weeks. At the start of treatment, he exhibited a low-level viremia with genotype 1b of the hepatitis C virus (HCV). After the first week of treatment with telaprevir, serum HCV RNA was undetectable, and negativity remained until the end of treatment. Moreover, he was evaluated as having a SVR after the post-treatment 24-week follow-up program. Two characteristics may explain the strong antiviral activity of telaprevir in the present case. First, although pre-treatment PCR-direct sequencing and cloning for the N-terminal in the NS3 region showed a protease inhibitor-resistant variant (T54A) in 1 of 32 independent clones, the T54A substitution has only a low-level resistance to protease inhibitors and his viral load was low. Second, when compared to a consequence sequence of 35 treatment-naïve patients with HCV genotype 1b, R130K and Q195K substitutions were unique to the present case. Although it is presently unknown whether the R130K and Q195K substitutions are related to SVR, this case suggests that long-term telaprevir monotherapy may be effective in CH-C patients with genotype 1 and a low viral load.     

A novel high throughput screening assay for HCV NS3 serine protease inhibitors

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.     

HCV NS3 N末端部分氨基酸缺失对NS3/NS3与NS3/NS4A分子间相互作用的影响

目的旨在弄清ＮＳ３参与分子间相互作用的确切区段，为研究针对ＮＳ３的抗ＨＣＶ寡肽小分子药物的设计提供依据。方法参照ＨＣＶ中国河北株序列设计ＮＳ３引物，将其Ｎ末端的前１５个和前３０个氨基酸分别缺失掉。然后用酵母双杂交系统检测ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用强度在缺失前后的变化，从而判明ＮＳ３Ｎ末端氨基酸在分子间相互作用中的意义。核苷酸序列分析采用ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍ３７３Ａ型自动测序仪。结果ＮＳ３Ｎ末端氨基酸缺失前后，ＮＳ３／ＮＳ３分子间及ＮＳ３／ＮＳ４Ａ分子间相互作用的强度相差有显著性（Ｐ＜０．０１），但缺失１５个氨基酸和缺失３０个氨基酸对上述相互作用强度的影响差异无显著性（Ｐ＞０．０５）。结论ＮＳ３Ｎ末端的１～３０个氨基酸在ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用中有一定意义，其Ｎ末端前１５个氨基酸（ＡＰＩＴＡＹＳＱＱＴＲＧＬＬＧ）对于分子间相互作用更为关键。本研究结果将为抗ＮＳ３丝氨酸蛋白酶活性的寡肽抑制物的研究打下基础，并为抗ＨＣＶ的寡肽小分子药物的设计提供依据     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.     

Complex formation between hepatitis C virus NS2 and NS3 proteins

Hepatitis C virus (HCV) NS2 and NS3 proteins as well as the NS3 protease cofactor NS4A are essential for the replication of the virus. The presence of in vivo heterodimeric complex between HCV NS2 and NS3 has been suggested by biochemical studies. Detailed characterization of the interactions between these viral proteins is of great importance for better understanding their role in viral replication cycle and represents attractive target for antiviral agents. In this study, we demonstrated in vivo interactions between HCV NS2 and NS3 proteins using an epitope tagging technique. For this purpose NS2, NS3 and NS4A were expressed in fusion with two different tags in Cos7 cells. Immunofluorescence analysis and co-immunoprecipitation with tag-specific antibodies revealed the existence of biologically important NS3/NS4A and NS3/NS2 complexes. Similar complexes were detected also in Huh7 cells infected with Semliki Forest virus vectors expressing NS2 and NS3 or NS23 precursor polyprotein. The formation of complex between NS2 and NS3 was found not to depend on whether the proteins were expressed individually or in form of common precursor. This observation suggests the existence of direct interaction between these two proteins that may have importance for the formation of the whole HCV replication complex.     

Development of robust in vitro serine protease assay based on recombinant Pakistani HCV NS3-4A protease

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 3 (predominant genotype in Pakistan) and they are poorly tolerated, highlighting the requirement of new therapeutics. HCV non-structural protein-3 (NS3) protease and helicase domains are essential for viral replication; they are highly conserved among various HCV strains. In the current study, we enrolled 56 HCV infected patients from various regions of Pakistan and determined their genotypes, ALT level and virus titer. We have cloned and sequenced NS3/NS4A from 4 of the HCV Serum samples. Nucleotide sequence alignment showed high level of identities among 3a genotypes. One of the samples (NCVI 01) showed unique amino acids substitutions, including R9Q, L332P, L354I, I605V and S622C. Three dimensional structures were determined and analyzed effect of substitutions on amino acids interactions. We further established fluorescence resonance energy transfer (FRET) based assays for detecting proteolytic activity of (NS3-4A) serine protease, using AnaSpec peptide, for high throughput screening (HTS) inhibitors against HCV. In future, this study could be of great interest in the development of HCV NS3 cell-based HTS FRET assay for genotype 3a and subsequent antiviral testing of drugs.     

A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.     

The acidic sequence of the NS4A cofactor regulates ATP hydrolysis by the HCV NS3 helicase

In flaviviruses and hepatitis C virus (HCV), the NS3 gene encodes the N-terminal protease (NS3pro) and the C-terminal helicase (NS3hel). In HCV, the downstream NS4A is required for the NS3pro activity and exhibits a conserved EFDEMEE motif. To identify the role of this motif, we compared the ATPase and helicase activities of NS3 alone with those of the NS3-NS4A constructs. Our results suggest that the EFDEMEE motif is essential for regulating the ATPase activity of NS3hel. It is likely that this motif interferes with the ATP-binding site of NS3hel. It is becoming clear that NS4A functions as a cofactor of both proteinase and helicase in HCV.     

聚合酶链反应诱导丙型肝炎病毒(HCV)蛋白酶活性位点的突变及突变HCVNS3/4a蛋白的表达

目的 以聚合酶链反应（PCR）突变方法诱导丙型肝炎病毒（HCV）蛋白酶活性位点ser1165的突变，获得全长非结构基因3（NS3）／4a的表达与纯化。方法 分别以NS3 N端正向引物与诱变反向引物，诱变正向引物与NS4a C端反向引物获得2个PCR产物，产物纯化后在新的PCR反应体系中加入以上2个PCR产物与NS3 N端正向引物、NS4a C端反向引物。再次PCR扩增突变模板，分别与野生型模板重组入表达载体pET26-Ub，转化大肠杆菌BL21（DE3）pCG1，诱导表达后经菌体裂解、纯清化、硫酸铵沉淀、DEAE－Sepharose、NTA纯化，Western blot分析表达蛋白的特异性及PCR诱导突变使HCV蛋白酶活性位点失活的作用。结果 获得诱导突变的模板，Western blot证实该突变可完全阻断对NS3丝氨酸蛋白酶与NS3螺旋酶间的切割，部分阻断了螺旋酶与NS4a间的切割，纯化后的HCV NS3／4a蛋白在SDS－PAGE胶上显示为双带。结论 PCR突变方法简便、有效，获得丝氨酸蛋白酶失活的NS3蛋白表达，NS3蛋白与NS4a蛋白以复合物形式存在。     

A wide range of NS3/4A protease catalytic efficiencies in HCV-infected individuals

The hepatitis C virus (HCV) NS3/4A protease acts as an antagonist of virus-induced interferon (IFN) regulatory factor (IRF)-3 activation and IFN-beta expression. The NS3/4A protease performs this function by cleaving Cardif and TRIF proteins to block retinoic-acid-inducible gen I (RIG-I) and toll-like receptor (TLR)-3 signaling, respectively. NS3/4A protease inhibition can prevent Cardif and/or TRIF inactivation during HCV infection, thereby maintaining the innate immune response. Thus, differences in NS3/4A protease catalytic efficiency could be related to viral pathogenicity. In this study, we analyzed the catalytic efficiency of the most abundant NS3/4A protease isolated from each of 12 individuals infected with HCV genotypes 1b, 1a, 3a, 4a or 4d. A diversity of NS3/4A protease catalytic efficiencies (up to a six-fold difference) was found in the analyzed samples. The genotype 1b NS3/4A proteases displayed the highest catalytic efficiencies. However, within this genotype up to three-fold differences were observed. Cross-genotypic interactions between the NS3 protease domain and the NS4A cofactor peptide were also investigated. Overall, catalytic efficiencies of NS3 proteases cross-interacting with NS4A cofactors from heterologous genotypes were as efficient as the homologous NS3/4A interactions. Of the 28 heterologous interactions tested, only 6 resulted in deleterious or nonfunctional enzymes. Nonfunctional interactions were not genotype-specific, suggesting that enhancement of NS3 catalytic efficiency by the NS4A cofactor depends on a few specific amino acid residues. Characterization of the proteolytic activities of individual NS3/4A proteases should provide clues for understanding HCV-host interactions, as well as assisting in the development of new classes of NS3/4A protease inhibitors.     

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.     

HCV NS3Ag: a reliable and clinically useful predictor of antiviral outcomes in genotype 1b hepatitis C virus-infected patients

Since hepatitis C virus (HCV) non-structural 3 (NS3) protease inhibitor (PI) combined with pegylated interferon/ribavirin (PR) has been approved for chronic HCV genotype (GT) 1b infection, a reliable and clinically useful predictor combining with serum HCV RNA to predict early virologic response, breakthrough, and relapse is important during HCV antiviral treatment. We evaluated the role of HCV NS3 antigen (HCV NS3Ag) on the prediction of virologic response in patients with HCV GT1b during PR or PR/simeprevir (triple) therapy. Three hundred patients were recruited, and HCV RNA and HCV NS3Ag were tested at baseline and weeks 2, 4, 12, 24, 48, and 72. NS3Ag and HCV RNA were significantly related (r² = 0.67) in the whole patient selection. The kinetic pattern of HCV RNA and HCV NS3Ag during triple treatment was similar. HCV NS3Ag levels in the triple group closely followed those of HCV RNA; the r² values were 0.756 (baseline), 0.837 (2 weeks), 0.989 (4 weeks), and 0.993 (12 weeks), respectively. For patients treated with PR, the positive and negative predictive values (PPVs and NPVs) for viral response were 96.31 % and 67.19 %, respectively, at week 4 by using the decrease of NS3Ag (dHCV NS3Ag) combined with HCV RNA. At week 12, the PPV was similar at 94.16 %, while the NPV reached 87.26 %. The PPV and NPV for the prediction of relapse and breakthrough were 90.6 % and 76.7 %, respectively. HCV NS3Ag is a valuable marker and could be a supplementary predictor of HCV RNA for the prediction of antiviral response, breakthrough, or relapse during HCV antiviral treatment.     

NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions

The gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminal region of nonstructural (NS) protein NS3 catalyzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B. Our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute together with the previously identified serine at position 1752 (S1752) the catalytic triad of the pestiviral NS3 serine protease. Interestingly, a mutant protease encompassing an exchange of the active site S1752 to threonine still showed residual activity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observed that the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4B site. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studies strongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable similarities observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggest a common ancestry between these enzymes and the capsid protease of Sindbis virus.     

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.     

PCR-Based In Vitro Synthesis of Hepatitis C Virus NS3 Protease for Rapid Phenotypic Resistance Testing of Protease Inhibitors

Protease inhibitors (PIs) targeting the hepatitis C virus (HCV) NS3 protease, such as telaprevir, have significantly improved the sustained virologic response (SVR) rates of HCV genotype 1 antiviral therapy. Given the expanding antiviral therapy regimen, fast HCV PI resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HCV PIs based on in vitro synthesis of patient-derived HCV NS3 protease and subsequent enzymatic testing in a fluorescent readout. The enzymatically active HCV NS3 proteases were synthesized from PCR-derived templates by an Escherichia coli S30 extract system. Tests of the protease genes with known mutations for telaprevir resistance showed that the phenotypic resistance test was fast, with a total turnaround time of <10 h, and was fully in agreement with the previous resistance results. The initial tests with 38 treatment-naive serum samples showed that the method was significantly less laborious and faster than currently available phenotypic resistance assays of HCV NS3 PIs.