Synergism between Tat and VP16 in trans-activation of HIV-1 LTR.
When tethered to heterologous DNA both Tat and VP16 can activate transcription from the HIV-1 LTR. To determine if they act by similar mechanisms, we constructed several hybrid effectors between Tat or VP16 and DNA-binding domains of GAL4 or LexA proteins. We tested these effectors on substituted reporter targets, which contained one to six GAL4 or LexA DNA-binding sites placed upstream of the HIV-1 promoter. Whereas Tat acted very inefficiently via DNA even with five DNA-binding sites, effects of VP16 were observed with a single DNA-binding site and increased with increasing number of sites. More importantly, effects of VP16 via DNA were synergistic with those of Tat via TAR RNA when both proteins were expressed simultaneously. We next created a tripartite fusion protein, which contained the GAL4 DNA-binding domain and activation domains of both Tat and VP16, which could be targeted to the HIV-1 LTR either via DNA or RNA. By introducing individual deleterious mutations into either Tat or VP16, we confirmed that effects of VP16 predominated via DNA whereas Tat but not VP16 acted via TAR RNA. Thus, Tat and VP16 act at different steps of the transcription process and increase expression from the HIV-1 LTR by different mechanisms.
Ghosh
S
S
Howard Hughes Medical Institute, University of California, San Francisco 94143-0724.
Selby
M J
MJ
Peterlin
B M
BM
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D016385:TATA Box; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099160:LexA Repressor Protein; D000099253:Trans-Activation Responsive RNA-Binding Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015696:Gene Products, tat; D017405:Herpes Simplex Virus Protein Vmw65; D009838:Oligodeoxyribonucleotides; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099160:LexA Repressor Protein; D000099253:Trans-Activation Responsive RNA-Binding Protein; C052130:GAL4 protein, S cerevisiae