HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR ELEMENT REVERTANT VIRUSES DEFINE RNA STRUCTURES REQUIRED FOR EFFICIENT VIRAL GENE-EXPRESSION AND REPLICATION

TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Ju rkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression an d replication. We previously demonstrated that viruses containing muta tions of the TAR RNA stem, bulge, or loop structures have 200- to 5,00 0-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jur kat cell lines infected with TAR element mutant viruses which spontane ously produced culture supernatants with wild-type-like levels of reve rse transcriptase activity, These viral supernatants were used to infe ct Jurkat cells, and following PCR amplification of the viral long ter minal repeats, their DNA sequences were analyzed. This analysis demons trated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR In gel retardation analysis, recombinant Tat prot ein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase re porter and HIV-1 proviral constructs and assayed following transfectio n of Jurkat cells. Constructs containing revertant TAR element mutatio ns were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary struc ture of TAR RNA sequences suggested that TAR RNA structures which diff ered from that of wild-type TAR were still capable of strong activatio n in response to Tat. These results further define critical sequences in TAR RNA that are required for fat activation. In addition, since TA R structures with lower free energy that preserve the loop and bulge s tructures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for fat activation.