A replicon-based shuttle vector system for assessing the phenotype of HCV NS5B polymerase genes isolated from patient populations

• Published in: Journal of virological methods Vol. 145; no. 2; pp. 137 - 145
• Main Authors: Middleton, Tim, He, Yupeng, Pilot-Matias, Tami, Tripathi, Rakesh, Lim, Ben Hock, Roth, Andrew, Chen, Chih-Ming, Koev, Gennadiy, Ng, Teresa I., Krishnan, Preethi, Pithawalla, Ron, Mondal, Rubina, Dekhtyar, Tatyana, Lu, Liangjun, Mo, Hongmei, Kati, Warren M., Molla, Akhteruzzaman
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.11.2007; Amsterdam Elsevier; New York, NY
• Subjects: Antiviral Agents - pharmacology; Biological and medical sciences; Drug Resistance, Viral; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; Genetic Vectors; Genotype; Hepacivirus - drug effects; Hepacivirus - enzymology; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis C - virology; Hepatitis C virus; Humans; Microbiology; NS5B; Phenotype; Plasmids; Replicon; RNA, Viral - isolation & purification; RNA-dependent RNA polymerase; RNA-Dependent RNA Polymerase - antagonists & inhibitors; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - isolation & purification; Shuttle vector; Techniques used in virology; Viral Nonstructural Proteins - antagonists & inhibitors; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - isolation & purification; Virology; Shuttle vector; Hepatitis C virus; NS5B; RNA-dependent RNA polymerase; Replicon; Human; Microbiology; Enzyme; Transferases; Method; RNA-directed RNA polymerase; Virology; Virus; Nucleotidyltransferases; Phenotype; Gene; Flaviviridae; Hepacivirus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.05.016

Hepatitis C virus (HCV) replicon-based shuttle vectors that permit phenotypes of NS5B polymerase genes from a large number of patient isolates to be rapidly assessed when transiently expressed in cultured cells were designed. When used to test responses to an inhibitor of HCV RNA-dependent RNA polymerase, IC 50 values for inhibition covered a several hundred-fold range among 47 patient samples tested. This observation highlights the variability that can be found by testing isolates derived from HCV-infected subjects. Partial suppression with a polymerase inhibitor of the most sensitive species permitted detection of minor quasispecies that were 7–200-fold more resistant than the bulk population in approximately half of the samples. Sequence analysis showed a wide range of amino acid changes not detected by conventional selection methods using laboratory-derived strains. This approach provides a means to assess variation in antiviral efficacy, and to predict possible responses in a clinical setting.