Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.     

Hepatitis C virus (HCV) NS5B nonnucleoside inhibitors specifically block single-stranded viral RNA synthesis catalyzed by HCV replication complexes in vitro

Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.     

Correlates of hepatitis C virus (HCV) RNA negativity among HCV-seropositive blood donors

BACKGROUND: Approximately 20 percent of persons infected with hepatitis C virus (HCV) clear viremia. Factors associated with resolution of viremia are not well defined. Implementation of routine nucleic acid testing (NAT) of blood donors has yielded a large data set for analysis of demographic correlates of resolved viremia. STUDY DESIGN AND METHODS: HCV antibody and NAT data, liver enzyme (alanine aminotransferase [ALT]) results, and donor demographic characteristics were compiled for 2,579,290 allogeneic donations given at five large blood centers after NAT implementation in 1999 through December 2001. Donation HCV RNA status was compared between first-time donors categorized by ALT levels, sex, age, race and/or ethnicity, country of birth, level of education, blood center location, and blood group, with chi-square tests and multivariable logistic regression methods. RESULTS: Of 35 confirmed-seropositive repeat donors, 19 (54.3%) tested negative for the presence of HCV RNA; there was no association between RNA status and preseroconversion intervals (p = 0.74). Of 2105 RIBA-positive, first-time donors, 402 (19.1%) tested negative for the presence of HCV RNA by NAT (presumptive resolved infections). There were significant differences in the frequency of RNA negativity among first-time donors categorized by ALT levels and by race and/or ethnicity. ALT levels were more likely to be elevated in RNA-positive, first-time donors (p < 0.0001). Viremia was less likely to resolve in Asian (8.2%) and black non-Hispanic (14.4%) donors than in white non-Hispanic (20.7%), Hispanic (22.1%), and other race and/or ethnicity (22.1%) donors (p = 0.02). No significant associations were found for age, sex, country of origin, level of education, blood type, and donor center location. CONCLUSION: These results confirm that the frequency of HCV RNA negativity among seropositive persons differs by race and/or ethnicity. Follow-up studies of donors with resolved viremia are warranted to further elucidate viral, immunologic, and genetic factors underlying spontaneous viral clearance.     

Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir

Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.     

Synergy of a hepatitis C virus (HCV) NS4A antagonist in combination with HCV protease and polymerase inhibitors

Rapid emergence of resistance to monotherapy with virus-specific inhibitors necessitates combination therapy. ACH-806 is a hepatitis C virus NS4A inhibitor with a novel mechanism of action and resistance pathway. This compound was synergistic with NS3 protease inhibitors and NS5B nucleoside and nonnucleoside polymerase inhibitors.     

Molecular mechanism of a thumb domain hepatitis C virus nonnucleoside RNA-dependent RNA polymerase inhibitor

A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique beta-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 microM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.     

丙型肝炎病毒RNA聚合酶在HepG-2细胞的稳定表达及活性分析

目的:探讨丙型肝炎病毒RNA聚合酶(NS5B)在肝癌细胞株HepG-2的稳定表达与活性.方法:Sca I酶切合有丙型肝炎病毒NS5B基因的重组表达载体pcDNA-5B,线性化的pcDNA-5B通过脂质体介导转染HepG-2细胞,G418筛选转染细胞.阳性细胞经基因组PCR、RT-PCR和Western Blot鉴定.体外RNA聚合酶试验和荧光素酶试验鉴定稳定表达NS5B蛋白的RNA聚合酶活性.结果:NS5B基因可整合到转染的HepG-2细胞的染色体上,并能有效转录和表达.体外RNA聚合酶试验和荧光素酶试验表明稳定表达的NS5B在体外和细胞内均具备RNA依赖的RNA聚合酶活性.结论:肝细胞HepG-2可以稳定表达丙型肝炎病毒NS5B,且具备RNA依赖的RNA聚合酶活性.     

Inhibition of hepatitis C replicon RNA synthesis by beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine: a specific inhibitor of hepatitis C virus replication

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.     

The role of hepatitis C virus (HCV) in mitochondrial DNA damage in HIV/HCV-coinfected individuals

Oxidative stress accompanying hepatitis C virus (HCV) infection seems to result in mitochondrial (mt) dysfunction. In HIV/HCV-coinfected individuals, HCV-related mt damage could be further enhanced and clinical manifestations of mt damage may appear, particularly following exposure to some antiretroviral drugs. Furthermore, when HCV medications are used together with certain antiretrovirals, the risk of developing mt adverse events may be particularly frequent, such as development of pancreatitis when ribavirin and didanosine are coadministered. The management of HIV/HCV-coinfected individuals needs to consider the high risk of mitochondria-associated toxicities in this population, which may significantly influence treatment decisions and therapeutic modalities.     

Increased detection of hepatitis C virus (HCV)-infected blood donors by a multiple-antigen HCV enzyme immunoassay

A new, multiple-antigen enzyme immunoassay (EIA-2) for hepatitis C virus (HCV) antibodies was evaluated in parallel with the previously available c100-3 HCV EIA (EIA-1) in 14,068 volunteer blood donors as well as in 25 cases of transfusion-associated hepatitis C for which recipient and donor samples were available. When compared to EIA-1, the EIA-2 was more sensitive in detecting HCV-infected blood donors. The EIA-2 detected an additional 1 in 1000 EIA-1-negative, surrogate marker-negative donors who were infected with HCV as demonstrated by polymerase chain reaction (PCR). The specificity of the EIA-2 was comparable to that of the EIA-1, but the two tests appear to detect different populations of false-positive donors. Recombinant immunoblot assay-indeterminate donors were detected five times more frequently by the EIA-2; PCR demonstrated that 21 percent of these donors were infected with HCV. The greater sensitivity of EIA-2 was also found in 25 transfusion recipients with non-A, non-B hepatitis; however, in 16 percent of these cases of posttransfusion HCV infection, the EIA-2 failed to detect an HCV-seropositive donor. These data indicate that EIA-2 testing will significantly reduce, but probably not eliminate, the risk of transfusion-associated HCV infection; we estimate this residual per-unit risk to be 1 in 2000 to 1 in 6000 units transfused. On a national level, it is projected that the replacement of the anti-HCV EIA-1 with the EIA-2 will initially prevent up to 40 additional cases of transfusion-associated hepatitis C per day.     

慢性丙型肝炎第一高变区抗体与HCV RNA病毒滴度相关性的研究

[目的]探讨慢性丙型肝炎患者第一高变区抗体与HCVRNA滴度的关系。[方法]采用计算机软件辅助分析确定27条HVR1基因序列，并克隆表达了系列重组HVR1抗原。应用27条不同的HVR1抗原包被酶联板，采用间接ELISA法测定慢性丙型肝炎患者样品HVR1抗体，并利用定量RT—PCR对病人血样完成HCVRNA测定，根据测定HCVRNA病毒滴度分为6组，10^2、10^3、10^4、10^5、10^6、10^7，每组6人，分别与27条不同的HVR1抗原进行反应，测定抗HVR1抗体，分别计算每组每种HVR1抗原的OD平均值。[结果]10^2、10^3、10^4组对27条HVR1抗体反应OD平均值均低于10^5、10^6、10^7组，而10^5组对27条HVR1抗体反应OD平值均最高。随着HCV RNA滴度的升高，27条HVR1抗体反应OD平均值略有下降。[结论]慢性丙型肝炎患者多种HCV第一高变区抗体与HCVRNA病毒滴度有一定的关系。     

Detection of GB virus C/hepatitis G virus RNA by reverse-transcription polymerase chain reaction.

Recently, a novel RNA virus, designated GB virus C or hepatitis G virus (GBV-C/HGV) has been identified which may possibly be associated with human hepatitis. In this study, the nucleotide sequences of the partial nonstructural 5 (NS5) gene of GBV-C/HGV derived from sera of eight Chinese patients were determined. The overall degree of nucleotide conservation and the existence of regional highly conserved sequences make this part of the genome suitable for the development of diagnostic reagents. On the basis of sequence analysis, two sets of oligonucleotide primers were designed to establish a nested polymerase chain reaction (PCR) for detection of GBV-C/HGV RNA. The efficacy of three PCR methods (first, one stage PCR, second, nested PCR with primers from the NS5 region designed according to the prototype sequence and the third, our newly developed PCR) was compared in 133 Chinese patients with liver disease. The positive rates of these three methods were 8.3%, 11.3% and 18.0% respectively. The specificity of our PCR detecting system was verified by sequencing and restriction fragment length polymorphism (RFLP). In conclusion, because of the heterogeneity and geographic distribution character of GBV-C/HGV, it is necessary to assess the sequence variation among Chinese patients infected with GBV-C/HGV. This may allow to identify GBV-C/HGV RNA with high sensitivity and specificity.     

DNA vaccine therapy for chronic hepatitis C virus (HCV) infection: immune control of a moving target

The use of DNA plasmids for DNA vaccination was first described in the early 1990s. DNA vaccinations were successful in small animal models but in larger animals and humans problems appeared. One major obstacle, effective delivery, has been partly overcome by new delivery techniques, such as transdermal delivery with the gene gun, and in vivo electroporation. We are entering a new era of DNA vaccination, where such techniques can be tested in humans. DNA vaccination may be a useful therapy for chronic hepatitis C virus (HCV) infections. Patients with these infections have a reduced T cell response to the invading virus. The genetic variability of HCV, its immunomodulatory properties and high replication rate contribute to chronicity. By providing the correct stimulus T cells may be activated to clear the infection. The vaccination is intended to induce a coordinated immune-based attack on the continuously moving HCV target. If effective, this should help in clearing the infection.     

Interferon-treated hepatitis C virus(HCV) patients with sustained biochemical response without eradication of HCV(asymptomatic HCV carrier)

Patients with hepatitis C virus(HCV) responding differently to interferon(IFN) therapy were speculated to have different incidence of disease progression to cirrhosis and of the development of hepatocellular carcinoma(HCC). However, the background and prognosis of the patients with sustained biochemical response without eradication of HCV (BR) (asymptomatic HCV carrier) has not been revealed so far. Review of recent studies suggest that the characteristics of the patients with BR are lower HCV RNA load, higher rate of HCV subtype-2 and lower score of liver fibrosis when compared with those with NR. The IFN therapy in patients who have not cleared HCV and showed normal ALT retards progression of fibrosis and reduces the incidence of cirrhosis and HCC.     

Evidence for persistent hepatitis C virus (HCV) infection in hemophiliacs.

Hepatitis C virus (HCV) is the major etiologic agent associated with non-A, non-B hepatitis. This study was designed to assess virologic and serologic markers in hemophiliacs exposed to non-heat-treated and/or virus-inactivated plasma derivatives. Serial bleeds from 48 hemophilic patients were analyzed for the presence of HCV viral RNA sequences as detected by polymerase chain reaction (PCR) and antibodies to structural (core) and nonstructural (C-100 and 33C) proteins by specific dot immunoblot assay. All patients exposed to non-heat-treated products, and four of six patients exposed only to virus inactivated products, had evidence of HCV infection. However, over the 5-yr study period, six exposed patients (13%) consistently lacked detectable anti-C-100 and seven (15%) lost this antibody. HCV viremia (PCR positive) was found in 91% of exposed patients, and was significantly more frequent in HIV seropositive hemophiliacs (P less than 0.05). Six patients had high antibody level to HCV and elevated ALT, but appeared to clear viremia. Four hemophiliacs were HCV seropositive but lacked detectable viremia. These data indicate that hemophiliacs remain persistently infected by HCV and that antibody to the core antigen of HCV is a reliable marker of this transfusion transmissible agent.