SP1 AND RELATED FACTORS FAIL TO INTERACT WITH THE NF-KAPPA-B-PROXIMALG C BOX IN THE LTR OF A REPLICATION COMPETENT, BRAIN-DERIVED STRAIN OF HIV-1 (YU-2)/

The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Spl transcription factor family whos e members can act as either activators of repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp bind ing sites with nuclear factors that are present in cell types that sup port HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electrophoretic mobility shift (E MS) competition analyses using oligonucleotides containing the sequenc es of each of the Sp1 sites of HIV-1 strain LAI, the NF-kappa B-proxim al Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Sp1 and related factors present in ly mphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Sp1 and two Sp3 isoforms were detected as the primary cellular constituents o f DNA-protein complexes formed with the NF-kappa B-proximal site. Only modest differences in Spl:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract . However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HI V-1 LAI Sp site III, a large difference in Spl and Sp3 binding was obs erved. To determine if naturally occurring and replication-competent s trains of HIV-1 contain base pair alterations within the Sp elements t hat affect the ability of the site to interact with Spl and related fa ctors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published seque nce of the YU-2 strain (a brain-derived macrophage tropic strain of HI V-1), displayed almost no Spl or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration , when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40-50% reduction in LTR activity in transiently transfe cted Turkat and U-373 MG cells. Overall, these results suggest that sp ecific G/C box sequence alterations present in the brain derived HIV-l variant YU-2, or possibly other brain-derived variants, may exhibit a ltered replication properties as a result of the low affinity of the N F-kappa B-proximal G/C box for members of the Sp transcription factor family.