Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein

Virology. 2000 Mar 15;268(2):251-63. doi: 10.1006/viro.2000.0194.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding, Competitive
  • Capsid / genetics*
  • Capsid Proteins*
  • Cell Line
  • Chloramphenicol O-Acetyltransferase / metabolism
  • DNA, Viral / analysis
  • DNA, Viral / metabolism
  • DNA-Binding Proteins / metabolism
  • Endogenous Retroviruses
  • Gene Expression Regulation, Viral
  • Gene Products, gag / genetics*
  • HIV Long Terminal Repeat / genetics*
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Plasmids / genetics
  • Transcription Factors / physiology
  • Transfection
  • Tumor Cells, Cultured
  • Viral Proteins*
  • Zinc Fingers / genetics
  • gag Gene Products, Human Immunodeficiency Virus

Substances

  • Capsid Proteins
  • DNA, Viral
  • DNA-Binding Proteins
  • Gene Products, gag
  • NCP7 protein, Human immunodeficiency virus 1
  • Transcription Factors
  • Viral Proteins
  • gag Gene Products, Human Immunodeficiency Virus
  • Chloramphenicol O-Acetyltransferase