A RECOMBINANT RETROVIRAL SYSTEM FOR RAPID IN-VIVO ANALYSIS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 SUSCEPTIBILITY TO REVERSE-TRANSCRIPTASE INHIBITORS

We have developed a new recombinant retroviral system in which a libra ry of infectious molecular clones of human immunodeficiency virus type 1 (HIV-1) is constructed with reverse transcriptase (RT) genes derive d from viral RNA sequences in plasma. HIV-1 RT is amplified from plasm a HIV-1 RNA by nested RT-PCR and cloned into a RT-defective HIV-1 prov iral vector (xxLAI-np), generating 10(3) to 10(4) recombinant proviral clones from each reaction. The bulk cloning products or individual mo lecular clones are transfected into MT-2 cells to generate infectious virus. The resultant viruses are assayed for drug susceptibility in CD 4(+) cell lines to determine either the dominant phenotype of the reco mbinant virus mixture or the phenotypes of the individual viral clones . DNA sequencing of the cloned RT genes can identify mutations associa ted with phenotypic resistance of clonal mixtures or individual clones . This method can be used to rapidly detect the in vivo emergence of H IV-1 quasispecies resistant to RT inhibitors.