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5th International Workshop on HIV Drug Resistance & Treatment Strategies4-8 June 2001, Scottsdale, Arizona |
New antiretroviral agents are being developed that target specific interactions between the HIV-1 envelope proteins and the cell surface receptors and co-receptors. We are using recombinant viruses in a single replication cycle assay to explore these interactions as well as the effects of inhibitory agents and natural ligands on viral infection. HIV-1 Env sequences (gp120, gp41) are amplified from HIV-1 (+) patient plasma or cell culture derived viral stocks and cloned as representative sequence libraries into an Env-expression plasmid. The Env-expression vector and an HIV-1 Gag-Pol expression vector that contains a luciferaseexpression cassette are introduced into 293 cells by co-transfection, which subsequently release replication- defective viral particles ‘pseudo-typed’ with the patient-derived HIV-1 Env proteins. The virus is used to infect target cells that express CD4 and CCR5 (R5) and/or CXCR4 (X4). Successful infection leads to the production of luciferase activity in the infected cells. We have tested primary and lab-adapted viruses representing R5, X4 and dual-tropic isolates. Co-receptor usage was evaluated by measuring the ability of the virus to infect either CD4-R5 or CD4–X4 cell lines. R5-tropic viruses were blocked at infection using R5 blocking agents and X4-tropic virus infection were inhibited using X4 blocking agents. The IC50 of the inhibitors varied based on the cell type used at infection and increased as the amount of cell surface co-receptor increased. Interestingly, in cell lines that express both R5 and X4 co-receptors, dual tropic viruses exhibited a complex pattern characterized by inhibition plateaus at 30–40% when tested in the presence of inhibitors. Susceptibility to the fusion inhibitor, DP178, varied substantially among DP178-naïve viruses (20–40-fold) and was independent of coreceptor tropism. The IC50 for DP178 differed significantly among the various cell lines tested and may be related to the amount of cell surface coreceptor. In summary, these studies indicate that defining X4, R5 co-receptor tropism and measuring virus entry/fusion inhibitor susceptibility are amenable to testing with recombinant virus assays.
PRESENTING AUTHOR: CJ Petropoulos
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