Activation of the human immunodeficiency virus long terminal repeat by varicella-zoster virus IE4 protein requires nuclear factor-kappaB 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 kappaB (NF-kappaB). 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-kappaB under the p50.p65 heterodimeric form and that no direct binding of IE4 to the kappaB sites could be detected. Both Western blot and immunofluorescence analyses suggested that the ability of IE4 to activate transcription through kappaB motives was not connected with its capacity to override the inhibitory activities of IkappaB-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-kappaB 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.
de Maisieres
P D
PD
Laboratory of Fundamental Virology and Immunology, Institute of Pathology, University of Liège, B-4000 Liège, Belgium.
Baudoux-Tebache
L
L
Merville
M P
MP
Rentier
B
B
Bours
V
V
Piette
J
J
D003545:Cysteine / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C085824:ORF4 protein, Human herpesvirus 3; D011498:Protein Precursors; D014764:Viral Proteins; D000072000:NF-KappaB Inhibitor alpha; D003545:Cysteine