Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein

Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h po stinfection. Since transcription factors with zinc fingers assist the trans criptional activity of both RNA polymerases II and III, we examined the eff ect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophor esis. Competition assays with cold probes revealed that the binding of NC a nd formation of a DNA-protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but: not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enh ances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappa B, Spl, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcr iption directed by the adenovirus major late promoter, however, is not sign ificantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 no or gag showed en hancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus con taining mutations in NC zinc-finger domains dramatically decreases the enha ncement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LT R and cooperatively enhances GTFs and NF-KB induced HIV-1 LTR basal-level a ctivity. NC may play the role of a nucleation protein, which binds to LTR a nd enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters. (C) 2 000 Academic Press.