TRANSCRIPTIONAL ACTIVATION OF MINIMAL HIV-I PROMOTER BY ORF-I PROTEIN EXPRESSED FROM THE SA/I-L FRAGMENT OF HUMAN HERPESVIRUS-6

The Sall-L fragment of human herpesvirus 6 (HHV-6) strain U1102 transformed rodent cells and transactivated the HIV-1 LTR 10- to 15-fold in both monkey fibroblasts and human T-lymphocytes. In this report, the Sall-L transactivator of the HIV-1 LTR was localized to ORF-I which codes for a protein of 357 amino acids. To determine if ORF-I required functional Spl binding sites or the TATA box element of HIV-1 LTR for transactivation, 5'-deletion mutants of the HIV-I LTR were employed. Plasmids pBS/Sall-L, pBS/Sall-L-SH, and pC6/ORF-1(S), a mammalian expression vector containing ORF-1, all transactivated a deletion mutant of HIV-1 LTR lacking functional Spl binding sites (CD-54). These studies demonstrate that transactivation occurred in the absence of Spl binding sites and required only a minimal HIV-1 promoter which contains the TATA box element. The specificity of the Sall-L transactivator for HIV-1 LTR was demonstrated by its inability to transactivate the human papillomavirus type 16 or 18 early promoters. The ORF-1 gene was cloned into and expressed from the pET17b bacterial expression vector. Purified ORF-I protein was obtained by ammonium sulfate precipitation, Mono-S chromatography, and anti-T7.Tag immunoaffinity chromatography. Transactivation of the HIV-l LTR by ORF-1 protein was demonstrated by electroporation studies in vivo and by transcription studies in vitro. To substantiate the putative biological role of ORF-1, pBS/Sall-L, pBS/Sall-L-SH, and pCG/ORF-1 all reactivated tat-defective HIV-1 provirus from latently infected cells expressing CD4. Thus, the data presented suggest that HHV-6 infection could have a cofactor role in the progression of AIDS. (C) 1994 Academic Press, Inc.