Two positive regulatory genes (tat and rev) of HIV are essential for viral replication. We propose to suppress HIV replication by manipulating the regulatory pathways governed by these two genes using a gene therapy approach. Two different approaches involving a) the use of dominant negative mutants that interfere with the activity of the wt tat and rev genes and b) competition inhibition of the activities of Tat and Rev by the respective target sequences, TAR and RRE, will be used to suppress the activities of these genes. We will attempt to construct a sequence- specific repressor of the tat gene by fusing the potential Tat sequences involved in interaction with TAR RNA with E. coli lacZ gene. In addition, attempts will also be made to isolate strong trans-dominant mutants of the tat gene by a genetic selection. Human 143tk- cells will be used to express the herpes simplex virus (HSV)-1 tk gene under the transcriptional control of the HIV-1 tat gene. Dominant negative mutants of the tat gene will be isolated from a pool of mutant tat genes expressed from an SV40- based vector by their ability to confer on 143tk+ cells resistance to the nucleoside analog Ganciclovir. Efficient dominant negative mutants of the rev gene will be isolated by introducing conservative changes in the 'effector' domain of Rev. The expression of dominant negative mutants will be maximized by placing them under the transcriptional control of strong promoters such as the cytomegalovirus immediate early gene promoter or a immunoglobulin gene mu-kappa hybrid promoter. In addition, the tat and rev mutant genes will also expressed from a single bicistronic mRNA. To increase the transnational efficiency of the mRNAs of these mutant genes, the sequences preceding the ATG codon will be converted to conform the sequences for maximal efficiency of transnational in mammalian cells. In the second approach, the activity of intracellular Tat and TAR-specific cellular factors will be competitively inhibited with vectors that express either monomeric or oligomeric forms of TAR sequences. Similarly, the activity of intracellular Rev will be inhibited with vectors expressing the HIV tat/rev 5'splice site and RRE. The ability of dominant negative tat or rev gene mutants and target sequence suppressors to inhibit HIV-1 replication will be determined in transient cotransfection experiments with proviral DNA and in T4 cell lines which stably express the various suppressors. The ability of these suppressors to protect human peripheral blood lymphocytes (PBL) against HIV-induced cytopathicity will also be determined by infection of PBL with retrovirus (M.MuLV) vectors expressing the suppressors and challenging such cells with various strains of HIV-1. These studies will form the basis for our long-term goal of suppressing HIV-mediated pathogenesis by 'intracellular immunization' or potential 'somatic gene therapy' approaches using viral vectors.
