RECOMBINANT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GENOMES WITH TAT UNCONSTRAINED BY OVERLAPPING READING FRAMES REVEAL RESIDUES IN TAT IMPORTANT FOR REPLICATION IN TISSUE-CULTURE

Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cyto pathicity. Extensive structure-function studies of Tat in subgenomic s ettings with point mutagenesis and transient transfection readouts hav e been performed. These reporter assays have defined certain amino aci d residues as being important for trans activation of reporter plasmid s. However, they have not directly addressed functions related to viru s replication. Here, we have studied Tat structure-function in the set ting of replicating viruses. We characterized mutations that emerged i n Tat during HIV-1 infections of T lymphocytes. To ensure that the sel ection pressure for change was directed toward protein function, we co nstructed HIV-1s in which the Tat reading frame was freed from constra ints exerted by overlapping with the reading frames of vpr, rev, and e nv. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tnt were observed from sequencing of 220 indepen dently isolated clones. Recloning of these changes into a pNL4-3 molec ular background allowed for the characterization of residues in Tat im portant for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activa tion of plasmid reporters were found to be relatively neutral. We conc lude that the structure-function of Tat in virus replication is incomp letely reflected by activity measurements based only on subgenomic tra nsient transfections.