CONSTRUCTION AND CHARACTERIZATION OF A POTENT HIV-2 TAT TRANSDOMINANTMUTANT PROTEIN

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) Tat p roteins Tat-1 and Tat-2 stimulate transcription of the viral long term inal repeat (LTR) sequences and are required for efficient viral repli cation. A class of mutant Tat proteins, termed ''transdominant mutants ,'' has been described that possesses relatively low transactivation a ctivity, yet is able to inhibit the function of wild-type Tat. These m utant proteins contain a nonfunctional TAR RNA-binding domain but appa rently retain a functional activation domain. A potential limitation f or therapeutic use of transdominant mutants described to date is their low but significant basal level of transactivation for the HIV-1 or H IV-2 LTRs. In order to make an improved transdominant mutant, we have constructed Tat-2 proteins that contain mutations in four contiguous a rginines at residues 81 to 84 in the RNA-binding domain. Using purifie d proteins and in vitro RNA-binding assays, we verified that these mut ant Tat-2 proteins are defective for TAR RNA binding. We also verified that these mutant Tat-2 proteins bind to a cellular protein kinase in vitro that we have previously shown to bind specifically to the Tat-1 and Tat-2 activation domain. Using plasmid cotransfection assays, we compared the phenotypes of these mutant Tat-2 proteins with the most p otent Tat-1 transdominant mutant described to date. One Tat-2 mutant, named ''R81-84A,'' was found to be equivalent to the Tat-1 mutant in a bility to inhibit wild-type Tat transactivation of HIV-1 and HIV-2 LTR s. Moreover, the R81-84A mutant possessed a significantly lower basal level of transactivation than the Tat-1 mutant. The R81-84A Tat-2 muta nt is therefore a promising reagent for future development as an anti- HIV agent. Additionally, our results suggest that wild-type Tat-2 tran sactivation of the HIV-2 LTR is especially sensitive to inhibition by transdominant mutants.