The roles of DEAD box helicases in the life cycle of HIV-1.

BACKGROUND: HIV/AIDS is the largest global public health problem; about 76 million people have been infected with HIV and 36 million people have already died. Existing antiviral treatment is successful but requires lifelong adherence and mostly targets viral factors. The virus mutates and evades both drugs and the human immune response. Cellular factors are potential therapeutic targets against HIV because the virus must conserve domains that interact with these cellular factors. Unlike many viruses HIV does not encode any helicases but it has been shown to use cellular DDX3. We screened the family of DEAD box helicases to seek other members as possible drug targets.

METHODS: We used a robust in-house siRNA knockdown technique to knockdown 59 cellular helicases. We measured viral production and infectivity using conventional transfection and infection assays in HeLa-M and TZM-bl cells. To determine whether the phenotypic results that we found were specific to depletion of the helicases and not due to off-target effects, we transfected rescue plasmids for each respective helicase.

FINDINGS: The library screen revealed five helicases that had not been previously identified as being associated with HIV-1 replication. We went on to study two of them in detail, the very closely related DDX5 and DDX17. We confirmed that knocking down DDX5 reduced HIV RNA and consequently viral production as measured by CA-p24 (capsid p24) and infectivity by two to three times compared with siControl-treated cells. Depletion of DDX17 reduced HIV-1 infectivity by five times and the extracellular (supernatant) CA-p24 by a similar reduction without affecting the intracellular HIV-1 Gag levels.

INTERPRETATION: Our results show that, despite their similarity and ability to form hetero (and homo) dimers, DDX5 and DDX17 are used by HIV in different phases of the lifecycle. DDX5 has a phenotype consistent with its involvement in viral transcriptional control. The phenotype of DDX17 knockdown suggests that it acts at a later timepoint after transcription. Detailed analysis of the exact processes affected by these two helicases is under further investigation.

FUNDING: Wellcome Trust.