A 4TH SP1 SITE IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 LONG TERMINAL REPEAT IS ESSENTIAL FOR NEGATIVE-SENSE TRANSCRIPTION

We report the discovery of a fourth Spl binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Spl IC), Localized to HXB2 nucleotides -433 to -441, This site is shown to bind Spl protein specifically in electroph oretic mobility shift assays. Spl protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Spl site, Mu tation of HIV-1 Spl IV in an HXB2-derived Long terminal repeat-chloram phenicol acetyltransferase reporter construct gave no significant chan ge in positive-sense transcription but abolished both basal and phorbo l myristate acetate-activated negative-sense transcription. Taken toge ther, the results further define the HIV-1 negative-sense promoter as an Spl-dependent, phorbol myristate acetate-responsive, and Tat-inhibi ted promoter initiating at HXB2 nucleotide -450.