Restriction factors are intracellular antiviral proteins that directly subvert viral replication processes.
Our research aims to identify antiviral restriction factors that target HIV in order to understand the molecular basis of virus control in patients and propose new paradigms for antiviral therapeutics. This has led to the recent identification of the cellular protein REAF (RPRD2) as a potent retrovirus targeting restriction factors, active in both cell lines and primary macrophages.
Yet little is known about REAF function. In cell lines REAF blocks HIV reverse transcription by unknown means, though gene ontology analysis suggests REAF likely has a role in the regulation of cellular nuclear RNA:DNA hybrids. However it is not known if the REAF driven viral replication block in macrophages occurs due to a direct inhibition of reverse transcription, or occurs due to the triggering of type-I interferon antiviral responses by REAF in response to viral nucleic acid – the latter being consistent with the role of macrophages as ‘immune sentinels’ of infection.
The first aim of this proposal will be to determine if nucleic acid target of REAF is RNA:DNA hybrids that are produced during HIV reverse transcription. This will be done both by nucleic acid immune precipitation with REAF, and by transfecting tagged nucleic acids probes into cells and determining localisation relative to REAF via confocal microscopy.
The second aim of this proposal is to determine if REAF directly blocks viral replication in macrophages, or if it acts as a pattern recognition receptor leading to antiviral cytokine responses. Macrophage cell lines and primary macrophages will be infected with HIV strains in the laboratory during REAF knockdown. Replication blocks will be measured by qPCR for viral nucleic acid species produced at each replication stage. Equally, induction of interferon responses will be measured by microarray to determine the up regulation of antiviral and inflammatory networks.
By determining the viral target recognised by REAF, and then by demonstrating the restriction mechanism, we will help understand how REAF may control HIV infection in patients, or to harness REAF expression as a novel antiviral therapy.
Dr Richard Sloan
Dr David Dockrell
Prof Jurgen Haas