Activation of the human immunodeficiency virus long terminal repeat by varicella-zoster virus IE4 protein requires nuclear factor-κB and involves both the amino-terminal and the carboxyl-terminal cysteine-rich region

Varicella zoster virus open reading frame 4-encoded protein (IE4) possesses transactivating properties for varicella-zoster virus genes as well as for those of heterologous viruses such as the human immunodeficiency virus type 1 (HIV-1). Mechanisms of HIV-1 LTR (long terminal repeat) transactivation were investigated in HeLa cells transiently transfected with an IE4 expression plasmid and a CAT reporter gene under the control of the HIV-1 LTR, These results demonstrated that IE4-mediated transactivation of the HIV-1 LTR in HeLa cells required transcription factor kappa B (NF-kappa B). Using the gel retardation assay, it was shown that transfection of the IE4 expression vector in HeLa cells was not associated with induction of NF-kappa B under the p50.p65 heterodimeric form and that no direct binding of IE4 to the kappa B sites could be detected. Both Western blot and immunofluorescence analyses suggested that the ability of IE4 to activate transcription through kappa B motives was not connected with its capacity to override the inhibitory activities of I kappa B-alpha or p105. Finally, in vitro protein-protein interactions involving IE4 and basal transcription factors such as TATA binding protein and transcription factor IIB were carried out. A direct interaction between IE4 and TATA-binding protein or transcription factor IIB components of the basal complex of transcription was evidenced, as well as binding to the p50 and p65 NF-kappa B subunits, Mutagenesis analysis of IE4 indicated that the COOH-terminal cysteine-rich and arginine-rich regions (residues 82-182) were critical for transactivation, whereas the first 81 amino acids appeared dispensable. Moreover, the arginine-rich region is required for the in vitro binding activity, whereas the COOH-terminal end did not appear essential.