A Survey of DDX21 Activity During Rev/RRE Complex Formation

J Mol Biol. 2018 Feb 16;430(4):537-553. doi: 10.1016/j.jmb.2017.06.023. Epub 2017 Jul 10.

Abstract

HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.

Keywords: DEAD-box protein; HIV-1; RNA structure; RRE; Rev.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / metabolism*
  • HIV Infections / metabolism*
  • HIV-1 / physiology*
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Maps*
  • rev Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • rev Gene Products, Human Immunodeficiency Virus
  • Adenosine Triphosphatases
  • DDX1 protein, human
  • DDX21 protein, human
  • DEAD-box RNA Helicases