ACTIVATION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS LONG TERMINAL REPEAT BY VARICELLA-ZOSTER VIRUS IE4 PROTEIN REQUIRES NUCLEAR FACTOR-KAPPA-B AND INVOLVES BOTH THE AMINO-TERMINAL AND THE CARBOXYL-TERMINAL CYSTEINE-RICH REGION

Varicella zoster virus open reading frame 4-encoded protein (IE4) poss esses transactivating properties for varicella-zoster virus genes as w ell as for those of heterologous viruses such as the human immunodefic iency virus type 1 (HIV-1). Mechanisms of HIV-1 LTR (long terminal rep eat) transactivation were investigated in HeLa cells transiently trans fected with an IE4 expression plasmid and a CAT reporter gene under th e control of the HIV-1 LTR, These results demonstrated that IE4-mediat ed transactivation of the HIV-1 LTR in HeLa cells required transcripti on factor kappa B (NF-kappa B). Using the gel retardation assay, it wa s shown that transfection of the IE4 expression vector in HeLa cells w as not associated with induction of NF-kappa B under the p50.p65 heter odimeric form and that no direct binding of IE4 to the kappa B sites c ould be detected. Both Western blot and immunofluorescence analyses su ggested that the ability of IE4 to activate transcription through kapp a B motives was not connected with its capacity to override the inhibi tory activities of I kappa B-alpha or p105. Finally, in vitro protein- protein interactions involving IE4 and basal transcription factors suc h as TATA binding protein and transcription factor IIB were carried ou t. A direct interaction between IE4 and TATA-binding protein or transc ription factor IIB components of the basal complex of transcription wa s evidenced, as well as binding to the p50 and p65 NF-kappa B subunits , Mutagenesis analysis of IE4 indicated that the COOH-terminal cystein e-rich and arginine-rich regions (residues 82-182) were critical for t ransactivation, whereas the first 81 amino acids appeared dispensable. Moreover, the arginine-rich region is required for the in vitro bindi ng activity, whereas the COOH-terminal end did not appear essential.