Abstract
HIV splicing involves five splice donor and eight splice acceptor sequences which, together with cryptic splice sites, generate over 100 mRNA species. Ninety percent of both partially spliced and fully spliced transcripts utilize the intrinsically weak A4/A5 3′ splice site cluster. We show that DDX17, but not its close paralog DDX5, specifically controls the usage of this splice acceptor group. In its absence, production of the viral envelope protein and other regulatory and accessory proteins is grossly reduced, while Vif, which uses the A1 splice acceptor, is unaffected. This is associated with a profound decrease in viral export from the cell. Loss of Vpu expression causing upregulation of cellular Tetherin compounds the phenotype. DDX17 utilizes distinct RNA binding motifs for its role in efficient HIV replication, and we identify RNA binding motifs essential for its role, while the Walker A, Walker B (DEAD), Q motif and the glycine doublet motif are all dispensable. We show that DDX17 interacts with SRSF1/SF2 and the heterodimeric auxiliary factor U2AF65/35, which are essential splicing factors in the generation of Rev and Env/Vpu transcripts.
Highlights
HIV differs from a number of other viruses that encode their own RNA helicases [1, 2], instead depending entirely on cellular helicases as essential cofactors for several stages of its replication cycle
DDX17 is essential for release of infectious HIV-1
We knocked down DDX17 by siRNA in cells transfected with a replication-competent proviral clone of HIV-1 pLAI and monitored the effects of DDX17 reduction upon viral infectivity
Summary
HIV differs from a number of other viruses that encode their own RNA helicases (such as the NS3 protein of hepatitis C) [1, 2], instead depending entirely on cellular helicases as essential cofactors for several stages of its replication cycle. The first of these to be identified was DDX3, which facilitates Rev-mediated export of unspliced and partially spliced viral transcripts [3]. Among those identified were two closely related DEAD box helicases, DDX5 and DDX17 [18]
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