Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex

Elife. 2016 Oct 12;5:e15910. doi: 10.7554/eLife.15910.

Abstract

HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.

Keywords: HDX; SAXS; SHAPE; X-ray crystallography; biochemistry; biophysics; integrative modeling; structural biology; virus.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cyclin T / chemistry*
  • Cyclin T / metabolism
  • Cyclin-Dependent Kinase 9 / chemistry*
  • Cyclin-Dependent Kinase 9 / metabolism
  • DNA, Viral / chemistry*
  • DNA, Viral / metabolism
  • Deuterium Exchange Measurement
  • HIV Long Terminal Repeat*
  • HIV-1 / genetics
  • Humans
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Proviruses / genetics
  • Repressor Proteins / chemistry*
  • Repressor Proteins / metabolism
  • Scattering, Small Angle
  • Transcription, Genetic
  • Transcriptional Elongation Factors / chemistry*
  • Transcriptional Elongation Factors / metabolism
  • tat Gene Products, Human Immunodeficiency Virus / chemistry*
  • tat Gene Products, Human Immunodeficiency Virus / metabolism

Substances

  • AFF4 protein, human
  • CCNT1 protein, human
  • Cyclin T
  • DNA, Viral
  • Repressor Proteins
  • Transcriptional Elongation Factors
  • tat Gene Products, Human Immunodeficiency Virus
  • CDK9 protein, human
  • Cyclin-Dependent Kinase 9