DIFFERENTIAL GROWTH-KINETICS ARE EXHIBITED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR MUTANTS

The human immunodeficiency virus type 1 (HIV-1) TAR element is critica l for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structu re containing both loop and bulge structures transcribed from TAR is t he major target for tat activation. Though transient assays have defin ed elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to gener ate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lin es which were capable of producing high titers of different viruses co ntaining TAR mutations. Viruses generated from these cell lines were u sed to infect both T-lymphocyte cell lines and peripheral blood mononu clear cells. Viruses containing TAR mutations in either the upper stem , the bulge, or the loop exhibited dramatically decreased HIV-1 gene e xpression and replication in all tell lines tested. However, we were a ble to isolate lymphoid cell lines which stably expressed gene product s from each of these TAR mutant viruses. Though the amounts of virus i n these cell lines were roughly equivalent, cells containing TAR mutan t viruses were extremely defective for gene expression compared with c ell lines containing wild-type virus. The magnitude of this decrease i n viral gene expression was much greater than previously seen in trans ient expression assays using HIV-1 long terminal repeat chloramphenico l acetyltransferase gene constructs. In contrast to the defects in vir al growth found in T-lymphocyte cell lines, several of the viruses con taining TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These re sults indicate that maintenance of the TAR element is critical for vir al gene expression and replication in all cell lines tested, though th e cell type which is infected is also a major determinant of the repli cation properties of TAR mutant viruses.