We will first verify the recently postulated block to elongation in the HIV-1 LTR, and then determine whether the tat gene product works by causing the anti-termination of transcription within the LTR, rather than through effects on RNA processing. This will be accomplished by performing nuclear runoff transcription assays using single-stranded probes and by determining the in vivo distribution of RNA polymerase II on the HIV-1 LTR, using a protein-DNA cross-linking method. These assays will be performed in mammalian cells co-transfected with the HIV-1 LTR and either wild-type or mutant tat genes. We will also define the sequences in cis necessary and sufficient for premature termination within the HIV-1 LTR co-injected with mutant and wild-type TAT gene into Xenopus oocytes. These experiments will determine if the proposed stem-loop structure within the LTR is necessary for the proposed termination/anti-termination of transcription, and if the site of tat binding is identical to the site of the elongation block. The Xenopus oocyte injection system will be used as the initial screen for these sequences. We will then determine if the cis sequences identified in the oocyte assay define the domain required for the termination/anti-termination of transcription within the HIV-1 LTR in mammalian cells. We will also determine if the tat gene product influences readthrough transcription of the endogenous c-myc, c-myb or c-fos genes which are known to be regulated by elongation blocks. The Xenopus oocyte system and a variety of human cell types will be used in this analysis. If tat does alleviate the elongation block to c-myc, we will also determine if tat expression is sufficient for the malignant transformation of Epstein Bar virus immortalized peripheral blood lymphocytes.
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