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Emmanouil Magiorkinis,
Dimitrios Paraskevis,
Maria G Detsika,
Liangjun Lu,
Gkikas Magiorkinis,
Marios Lazanas,
Stijn Imbrechts,
Kristel Van Laethem,
Anne-Mieke Vandamme, Tami Pilot-Matias,
Akhteruzzaman Molla,
Ricardo J Camacho,
Angelos Hatzakis
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ABSTRACT: HIV drug resistance is a multifactorial phenomenon and constitutes a major concern as it results in therapy failure. The aim of this study was to assess the impact of an amino acid insertion identified at position 33 of the protease gene, derived from samples from three patients under lopinavir therapy, on viral fitness and protease inhibitor (PI) resistance. Successive samples were available from one of the patients for genotypic and phenotypic testing in order to investigate the role of this insertion. The patient had been pretreated with various antiretroviral drugs and showed poor virological response from the point of the acquisition of the mutation onward. The insertion was acquired in the context of a number of other PI mutations and was stable following acquisition. Phenotypic testing revealed reduced susceptibility to various PIs and a reduction of the replicative capacity (RC) of the virus. In the presence of the insertion alone, a decrease of the RC was observed, which seemed to be compensated by the presence of other mutations. The L33ins might have a potential role in PI resistance pathways but further investigation in a larger number of clinical samples is required in order to elucidate this resistance mechanism.
AIDS research and human retroviruses 03/2011; 27(11):1223-9. · 2.18 Impact Factor
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Ann D Kwong,
Isabel Najera,
Jill Bechtel,
Scott Bowden,
Joseph Fitzgibbon,
Patrick Harrington,
Dale Kempf,
Tara L Kieffer,
Diana Koletzki,
George Kukolj, [......],
Chris Petropoulos,
Gaston Picchio,
Robert Ralston,
Jacqueline D Reeves,
Robert T Schooley,
Scott Seiwert,
David Standring,
Lieven Stuyver,
James Sullivan,
Veronica Miller
Gastroenterology 01/2011; 140(3):755-60. · 11.68 Impact Factor
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ABSTRACT: Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir.
Antimicrobial Agents and Chemotherapy 11/2010; 54(11):4903-6. · 4.84 Impact Factor
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Tim Middleton,
Yupeng He, Tami Pilot-Matias,
Rakesh Tripathi,
Ben Hock Lim,
Andrew Roth,
Chih-Ming Chen,
Gennadiy Koev,
Teresa I Ng,
Preethi Krishnan,
Ron Pithawalla,
Rubina Mondal,
Tatyana Dekhtyar,
Liangjun Lu,
Hongmei Mo,
Warren M Kati,
Akhteruzzaman Molla
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ABSTRACT: 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.
Journal of Virological Methods 12/2007; 145(2):137-45. · 2.01 Impact Factor
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Liangjun Lu,
Tatyana Dekhtyar,
Sherie Masse,
Ron Pithawalla,
Preethi Krishnan,
Wenping He,
Teresa Ng,
Gennadiy Koev,
Kent Stewart,
Dan Larson,
Todd Bosse,
Rolf Wagner, Tami Pilot-Matias,
Hongmei Mo,
Akhteruzumman Molla
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ABSTRACT: Compound A-837093, a non-nucleoside HCV RNA-dependent RNA polymerase inhibitor, displayed nanomolar potencies against HCV genotypes 1a and 1b replicons. It also exhibited an excellent metabolic profile and achieved high plasma and liver concentrations in animals. In order to characterize the development of resistance to this anti-HCV agent, HCV subgenomic 1b strain N replicon cells were cultured in the presence of A-837093 with G418. Mutations S368A, Y448H, G554D, Y555C, and D559G in the NS5B polymerase gene were identified that led to substantial decreases in the susceptibilities of 1b genotype replicons to the inhibitor A-837093. However, the resistant mutants remained susceptible to HCV protease inhibitor BILN-2061 and alpha interferon as well as to a different class of non-nucleoside HCV polymerase inhibitor. In addition, each single resistant mutation identified significantly reduced the replication capacity of mutant compared to wild-type replicon. These findings provide a strategic guide for the future development of non-nucleoside inhibitors of HCV NS5B polymerase.
Antiviral Research 11/2007; 76(1):93-7. · 4.30 Impact Factor
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Teresa I Ng,
Hongmei Mo, Tami Pilot-Matias,
Yupeng He,
Gennadiy Koev,
Preethi Krishnan,
Rubina Mondal,
Ron Pithawalla,
Wenping He,
Tanya Dekhtyar,
Jeremy Packer,
Mark Schurdak,
Akhteruzzaman Molla
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ABSTRACT: Hepatitis C virus (HCV) replication is highly dependent on host cell factors. Identification of these host factors not only facilitates understanding of the biology of HCV infection but also enables the discovery of novel targets for anti-HCV therapy. To identify host genes important for HCV RNA replication, we screened a library of small interfering RNA (siRNA) that targets approximately 4,000 human genes in Huh7-derived EN5-3 cells harboring an HCV subgenomic replicon with the nonstructural region NS3-NS5B from the 1b-N strain. Nine cellular genes that potentially regulate HCV replication were identified in this screen. Silencing of these genes resulted in inhibition of HCV replication by more than 60% and exhibited minimal toxicity. Knockdown of host gene expression by these siRNAs was confirmed at the RNA level and, in some instances, at the protein level. The level of siRNA silencing of these host genes correlated well with inhibition of HCV. These genes included those that encoded a G-protein coupled receptor (TBXA2R), a membrane protein (LTbeta), an adapter protein (TRAF2), 2 transcription factors (RelA and NFkappaB2), 2 protein kinases (MKK7 and SNARK), and 2 closely related transporter proteins (SLC12A4 and SLC12A5). Of interest, some of these genes are members of the tumor necrosis factor/lymphotoxin signaling pathway. CONCLUSION: Findings of this study may provide important information for understanding HCV replication. In addition, these cellular genes may constitute a novel set of targets for HCV antiviral therapy.
Hepatology 07/2007; 45(6):1413-21. · 11.66 Impact Factor
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Rakesh L Tripathi,
Preethi Krishnan,
Yupeng He,
Tim Middleton, Tami Pilot-Matias,
Chih-Ming Chen,
Daryl T Y Lau,
Stanley M Lemon,
Hongmei Mo,
Warren Kati,
Akhteruzzaman Molla
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ABSTRACT: A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from "quasispecies" pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A-5B genes were constructed with the S2204I "adaptive" mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A-5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-alpha (IFN-alpha) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC(50) values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A-5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-alpha and to small molecule inhibitors.
Antiviral Research 02/2007; 73(1):40-9. · 4.30 Impact Factor
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Hongmei Mo,
Liangjun Lu, Tami Pilot-Matias,
Ron Pithawalla,
Rubina Mondal,
Sherie Masse,
Tatyana Dekhtyar,
Teresa Ng,
Gennadiy Koev,
Vincent Stoll,
Kent D Stewart,
John Pratt,
Pam Donner,
Todd Rockway,
Clarence Maring,
Akhteruzzaman Molla
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ABSTRACT: 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.
Antimicrobial Agents and Chemotherapy 11/2005; 49(10):4305-14. · 4.84 Impact Factor
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ABSTRACT: The Hepatitis C (HCV) replicon system is a useful tool for the high-volume screening of inhibitors of HCV replication. In this report, a cell-based assay has been described, which monitors the inhibition of HCV genotypes 1a and 1b as well as cytotoxicity, from a single well of a 96-well plate. A mixture of two stable replicon cell lines was used: one containing a 1a-H77 replicon expressing a firefly luciferase reporter, and the other one containing a 1b-N replicon with a secreted alkaline phosphatase reporter, thus allowing us to monitor replication of two HCV genotypes in the same well. Cytotoxicity was measured using the Resazurin cytotoxicity assay. The assay was validated with known HCV inhibitors and showed that the antiviral activity and cytotoxicity of compounds were reproducibly measured under screening conditions. It was also showed that the assay's signal-to-noise ratio and Z′ coefficient were suitable for high-throughput screening. A panel of HCV inhibitors showed a good correlation between EC50 and TD50 values for 1a and 1b replicon activity and cytotoxicity measured using either a single replicon format or mixed replicon format. Thus, the use of this mixed replicon format provides an economical method for simultaneous measurement of compound activity against two HCV genotypes as well as cytotoxicity, thereby reducing cost of reagents and labor as well as improving throughput.
Antiviral Research.
