HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log 10 HCV copies/g total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10-to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four-to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.Hepatitis C virus (HCV) is an enveloped, positive-sense, single-stranded RNA virus of approximately 9.6 kb that possesses an RNA-dependent RNA polymerase (RdRp), NS5B. Like that in many RNA viruses, this RNA replicase lacks a proofreading mechanism. The mutation rate of the HCV RdRp is estimated to be 10
Hepatitis C virus (HCV) infection is the cause of significant long-term morbidity and mortality. While often asymptomatic, the majority of HCV infections result in chronic hepatitis that can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. It is estimated that about 170 million people worldwide, approximately 3% of the world's population, are infected with HCV (45). Presently, there is no specific antiviral agent directed against HCV and no vaccine for prevention of hepatitis C infection. The current approved treatments, interferon monotherapy or interferon in combination with ribavirin, have limited benefits. Interferon alone achieves a sustained viral response in only 10 to 20% of patients (32), while the recommended therapy of pegylated interferon in combination with ribavirin results in a sustained viral response in 54% of patients (23). The response rate is lower in patients who are infected with HCV genotype 1b (ϳ34%) (23,47). Adverse side effects, such as severe flu-like symptoms, depression, psychoses, and anemia, are associated with these treatments, causing approximately 20% of patients to discontinue therapy (13,17,33). Consequently, there is an urgent need for the development of an HCV-specific antiviral that is more effective, less toxic, and easier to administer than the present therapy.HCV, a member of the Flaviviridae family, is a positivesense, single-stranded RNA virus with a genome size of ϳ9.4 kb (26, 39). The genome RNA encodes a polyprotein of 3,010 to 3,011 amino acid residues in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein is processed by host and viral proteases (18,34). The nonstructural protein 5B (NS5B) is a virus-encoded RNA-dependent RNA polymerase (RdRp) that is responsible for replication of the viral RNA genome. A functional counterpart of NS5B does not exist in mammalian cells. For this reason, an inhibitor of NS5B could serve as an effective and selective agent for treating HCV infection.The enzymatic activity of the NS5B enzyme in vitro has been extensively characterized (9,19,21,40). Both the full-length and carboxyl-terminally truncated forms of NS5B have been shown to be functionally active in the presence of HCV or exogenous RNA templates. Although HCV replicates inefficiently in cell culture, viral replication, including the activity of the NS5B polymerase, can be studied in a cell culture system
Background Identifying Streptococcus pneumoniae serotypes by urinary antigen detection (UAD) assay is the most sensitive way to evaluate the epidemiology of nonbacteremic community-acquired pneumonia (CAP). We first described a UAD assay to detect the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, covered by the licensed 13-valent S. pneumoniae conjugate vaccine. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) in individuals with radiographically confirmed CAP. Methods The specificity of the UAD-2 assay was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies, using Luminex technology. Assay qualification was used to assess accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by nonparametric statistical evaluation of urine samples from individuals without pneumococcal disease. The sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation, using urine samples obtained from a large study that measured the proportion of radiographically confirmed CAP caused by S. pneumoniae serotypes in hospitalized US adults. Results The UAD-2 assay was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a reference standard of bacteremic pneumonia. In addition, the UAD-2 assay identified a S. pneumoniae serotype in 3.72% of nonbacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the proportion of pneumonias with a UAD-2 serotype was 4.33%. Conclusions The qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic S. pneumoniae CAP and for assessing the efficacy of future pneumococcal conjugate vaccines that are under development.
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 -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 M. 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.A wealth of literature has documented that RNA viruses or viruses that use RNA replicative intermediates have high degrees of genetic variability (5, 15). The high mutation rates found in these organisms are largely due to the lack of proofreading mechanisms and/or a high recombination frequency during cellular DNA replication. A DNA-dependent DNA polymerase complex allows an error rate of 10 Ϫ9 mutations/ nucleotide, whereas the error rates for an RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase (RdRp), such as those in human immunodeficiency virus (HIV) and hepatitis C virus (HCV), respectively, are on the order of 10 Ϫ4 to 10 Ϫ5 mutations/nucleotide (35, 37). The robust viral replication that leads to a large population size during viral infection in these RNA viruses and retroviruses contributes to the generation of quasispecies in infected patients (7,12,13).During chemotherapy, the high rates of viral replication and the high frequency of mutation lead to the rapid generation of drug-resistant mutants. In the case of HIV, numerous mutations have been identified in patients treated with protease inhibitors as well as nucleoside and nonnucleoside reverse transcriptase inhibitors. The emergence of resistant viruses becomes one of the biggest challenges in developing effective antiviral therapies to eradicate these ...
BackgroundIdentifying Streptococcus pneumoniae (Sp) serotypes by urinary antigen detection assay (UAD) is the most sensitive and specific way to evaluate the changing epidemiology of non-bacteremic community-acquired pneumonia (CAP) and efficacy of pneumococcal vaccines. We first described an UAD to detect the Sp serotypes 1,-3,-4,-5,-6A,-6B,-7F,-9V,-14,-18C,-19A,-19F,-23F covered by the 13-valent Sp conjugate vaccine PCV13. To assess the pneumococcal disease burden of additional serotypes, a UAD-2 assay was developed to diagnose 11 additional Sp serotypes (-2,-8,-9N,-10A,-11A,-12F,-15B,-17F,-20,-22F,-33F).MethodsUAD-2 specificity was achieved by capturing highly purified pneumococcal polysaccharides with serotype-specific monoclonal antibodies using Luminex technology. Assay qualification assessed accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by non-parametric statistical evaluation of urine samples from individuals without pneumococcal disease. Clinical sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation.ResultsThe UAD-2 was shown to be specific and reproducible. Clinical validation using urine samples from invasive disease patients demonstrated assay sensitivity and specificity of 92.2% and 95.9%, respectively compared with a gold standard of isolating and typing (by Quellung) Sp bacteria from patient samples. Analysis of 11,087 CAP patients showed a UAD-2 and UAD-1 serotype prevalence of 4.33% and 4.60%, respectively (bacteremic and non-bacteremic CAP combined).ConclusionThe qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic Sp CAP as well as assessing vaccine efficacy of future pneumococcal conjugate vaccines.Disclosures All authors: No reported disclosures.
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