Patent ID: 6558923
Filing Date: 2003-05-06
Classification: C12Q

Abstract:
An in vitro, single cycle, recombinant virus assay (RVA) for determining HIV susceptibility to protease inhibitor, wherein the method comprises:(A) transfecting at least one eukaryotic cell with, (1) amplified HIV protease sequences of an HIV virus to be assayed for resistance to the protease inhibitor; (2) an HIV envelope defective molecular clone, wherein the molecular clone has a complete deletion of its protease coding sequence of pol, two deletions in its envelope gene (env), and a deletion of part, but not all, of its gag gene; and (3) a plasmid containing VSV-G envelope coding sequence under the control of a promoter for phenotypic complementation of the HIV, envelope defective, molecular clone; (B) culturing the resulting transfected cells to repair the protease coding sequence by homologous recombination between the HIV molecular clone and the amplified HIV protease sequences, and to produce a testable stock of infectious particles by coexpression of the protease-repaired HIV molecular clone and the VSV-G envelope coding sequence; (C) contacting cells of the culture from step (B) with the protease inhibitor; (D) infecting indicator cells containing an indicator gene with the testable stock of infectious particles from step (C), without amplification of the infectious particles prior to infecting the indicator cells; (E) expressing the indicator gene in the indicator cells from step (D); and (F) determining accumulation of indicator gene product as a measure of inhibition of HIV replication by the protease inhibitor.