A Survey of DDX21 Activity During Rev/RRE Complex Formation. Issue 4 (16th February 2018) - Hammond, John A.
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. Graphical abstract: Highlights: DDX21 has been implicated in HIV-1 Rev-dependent nuclear export. DDX21 is an RNA-dependent ATPase, with both ATP-dependent and ATP-independent helicase activities. DDX21 ATP-dependent activities (ATPase and RNA helicase) are inhibited by the presence of Rev, and this inhibition is dependent on Rev binding to RNA substrates. DEAD-box proteins bind to Rev through their conserved N-terminal DEAD domain, and Rev through its NIS domain. DDX21 is able to stimulate Rev binding to the RRE in a manner similar to that described for DDX1, while DDX3 cannot.