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 postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect 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 electrophoresis. Competition assays with cold probes revealed that the binding of NC and 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 enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappaB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement 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 LTR and cooperatively enhances GTFs and NF-kappaB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters.
Zhang
J L
JL
Division of Infectious Diseases, Charles A. Dana Research Institute, Boston, Massachusetts, USA.
Sharma
P L
PL
Crumpacker
C S
CS
D001483:Base Sequence; D001667:Binding, Competitive; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D020077:Endogenous Retroviruses; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000235:genetics*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins; D016335:Zinc Fingers / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D014157:Transcription Factors; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase