Abstract
BackgroundHIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein–protein interactions and RNA and ATP binding.ResultsWe demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable.ConclusionDDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.
Highlights
Human immunodeficiency virus (HIV)-1 does not encode a helicase and hijacks those of the cell for efficient replication
Cellular positive transcription elongation factor b (P-TEFb) is stored mainly in the inactive form sequestered with HEXIM1/2 bound to the 7SK snRNP complex, Human immunodeficiency virus type 1 (HIV-1) Tat competes with HEXIM1/2 to disrupt its binding on P-TEFb [23] detailed understanding of the molecular mechanisms behind Tat dislodging HEXIM1/2 and successfully preventing HEXIM1/2 from binding to free P-TEFb has been lacking
Our co-immunoprecipitation data reveal that DDX5 can interact with both Tat and HEXIM1 within the cell and that it has a significant potentiating effect when coexpressed with a Tat expressing plasmid in TZM-bl cells (Fig. 3a–e) and in Hela cells (Additional file 3: Fig. S3a, b) providing a plausible scenario that DDX5/Tat interacts with the 7SK snRNP releasing P-TEFb and sequestering HEXIM to block its inhibitory effect
Summary
HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. DEAD box RNA helicases are ubiquitous cellular proteins with protean functions. They derive their name from the first letter amino acid sequence of their conserved Walker B motif. Human immunodeficiency virus type 1 (HIV-1) does not, instead utilizing cellular helicases as essential cofactors at 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 [5]. DDX1 was shown to be a cofactor of Rev [6] while RNA helicase A (RHA) promotes HIV-1 reverse transcriptase, transcription and translation [6,7,8,9,10]
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