Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat

PLoS Pathog. 2010 Oct 14;6(10):e1001152. doi: 10.1371/journal.ppat.1001152.

Abstract

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Flanking Region / genetics
  • Base Sequence
  • Binding Sites / genetics
  • Cells, Cultured
  • Gene Expression Regulation, Viral
  • HIV-1 / genetics
  • HIV-1 / metabolism*
  • HeLa Cells
  • Humans
  • Models, Biological
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Binding
  • Protein Multimerization
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • RNA-Binding Proteins / metabolism
  • Transcription Factors
  • Transcriptional Activation / genetics
  • tat Gene Products, Human Immunodeficiency Virus / metabolism
  • tat Gene Products, Human Immunodeficiency Virus / physiology*

Substances

  • HEXIM1 protein, human
  • RNA, Small Nuclear
  • RNA-Binding Proteins
  • Transcription Factors
  • tat Gene Products, Human Immunodeficiency Virus
  • Positive Transcriptional Elongation Factor B