4th International Workshop on HIV Drug Resistance & Treatment Strategies Sitges, Spain, 12–16 June 2000

SENSITIVITY OF DETECTION OF MINORITY SPECIES IN A SINGLE CYCLE REPLICATION ASSAY IS INFLUENCED BY THE BIOCHEMICAL MECHANISM OF RESISTANCE: INSIGHT INTO THE MECHANISM OF NRTI-RESISTANCE AND CROSS-RESISTANCE IN PATIENT SAMPLES

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):16 (Abstract 18

MM Sartoris, W Huang, C Petropoulos and JM Whitcomb
ViroLogic, Inc., South San Francisco, Calif., USA

Co-transfection of cells with a mixture of recombinant HIV-1 vectors derived from NRTI-sensitive and NRTI- resistant viruses results in the production of virions that contain both drug-sensitive and drug-resistant RT molecules within the same virus particle. After such ‘phenotypically mixed’ virus particles infect new cells, the drug-sensitive and drug-resistant RT molecules compete during the RT step of viral replication. During reverse transcription, each RT molecule cycles on and off of the template/primer complex at a rate determined primarily by the processivity of the enzyme. In doing so, both sensitive and resistant RT molecules have an opportunity to contribute to reverse transcription of the viral genome. In the presence of RT inhibitors, this shared contribution is reflected in a characteristic phenotypic susceptibility profile. We have evaluated the phenotypic drug susceptibility profiles of mixtures of recombinant viral vectors derived from NRTI-resistant and NRTI-sensitive HIV 1. The phenotypic profiles of mixtures varied depending on the NRTI-mutations present in the resistant component. When tested against zidovudine, incremental mixtures comprised of a zidovudine-sensitive component and a zidovudine-resistant component containing various combinations of mutations at M41L, D67N, K70R, L210W, T215Y/F and K219Q/E, or the T69SSX insertion complex exhibited distinct intermediate zidovudine susceptibility profiles (mixing phenotype). Conversely, incremental mixtures comprised of a zidovudine-sensitive component and a zidovudine-resistant component containing Q151M, A62V, V77I and F116Y exhibited wild-type zidovudine susceptibility profiles (non-mixing phenotype). When tested against lamivudine, incremental mixtures comprised a sensitive component and a resistant component containing M41L, E44D, V118I, T215Y or the T69SSX insertion complex displayed a lamivudine mixing phenotype. Mixtures containing M184V/I displayed a dampened lamivudine mixing phenotype. In contrast, incremental mixtures comprised of a sensitive component and a resistant component containing the Q151M complex displayed a lamivudine non- mixing phenotype. We hypothesize that the degree to which individual viral components exhibit a mixing phenotype is determined, at least in part, by the mechanism of resistance to the antiviral agent. We will present data supporting a model that explains mixing patterns based on proposed mechanisms of NRTI resistance (in other words phosphorolysis versus dNTP discrimination). The model also addresses the contribution of factors that are thought to influence viral replication capacity, or fitness (for example RT processivity). The model further provides a testable hypothesis to explain NRTI cross-resistance as a measure of the ability of different clusters of mutations to remove different chain terminators, via phosphorolysis.

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2000-06-12
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