CORDIS - EU research results

The role of centrosomes in HIV cytoplasmic transport

Final Report Summary - HIV TRAFFIC CONTROL (The role of centrosomes in HIV cytoplasmic transport)

The focus of the research project is to identify cellular factors influencing trafficking of the HIV genomic RNA (gRNA) from the perinuclear regions where it is translated into structural proteins (Gag, Gag-Pol) to the plasma membrane virus assembly site. Trafficking of these viral components has been suggested to occur via centrosomes and microtubules, but little is known about this critical virus-host cell interaction. We thus investigated co-localisation of gRNA and Gag with centrosomes using confocal microscopy. Despite a thorough optimisation, co-localisation of the viral components with the centrosome was not detected. The centrosomal localisation of the gRNA and Gag may be transient and could be missed in the steady state experiment. To extend the duration of the HIV-centrosome interaction and improve its detection, we aimed to inhibit downstream trafficking of the viral complexes. This could be achieved by knocking down a motor protein essential for transporting the HIV gRNA complex away from the centrosome.

We set up a genetic screening system to identify this hitherto unknown motor protein. Suitable cell lines were transfected with HIV infectious DNA and a library of pooled siRNAs against the microtubule-associated motor proteins. Rank product analysis revealed the statistically most significant hits. The results show that some siRNAs reduce and others enhance particle production, with cell and process specific effects. We selected five candidates for further investigation. We aimed to confirm the specificity of the siRNA-mediated knock down to rule out off-target effects influencing HIV particle production. In the case of a specific, on-target effect expression of siRNA-resistant rescue plasmid should restore virus particle production in siRNA-treated cells. We did not observe a rescue effect for any of the selected candidates. However, expression levels of the rescue ORFs were low in HIV-producing cells, indicative of technical obstacles. The siRNA-resistant plasmids expressed well in the absence of HIV production, confirming that the expression cassettes were functional. Attempts to assess the co-localisation of the motor proteins and viral components with confocal microscopy failed due to antibodies of inadequate specificity.

We further focused on KIF7, the top-ranked candidate from the siRNA screens. Total Gag production (virus-free and -associated) was reduced upon KIF7 knock down, suggesting that KIF7 plays a role in intracellular Gag accumulation. A dominant negative (DN) KIF7 mutant was constructed in which the microtubule-interacting domain was removed, leaving the cargo-interacting domain intact. Expression of this DN mutant should block microtubular transport of any KIF7 cargo, but did not inhibit virus production reproducibly. Lack of suitable antibodies against KIF7 again hampered analysis of DN KIF7 expression levels and therefore we cannot draw firm conclusions on the role of KIF7 in the HIV life cycle. Currently, we are constructing Myc-tagged versions of the wild type and DN KIF7 proteins to assess their microtubule association and potential co-localisation with HIV components.

Over 30 million people are infected with HIV worldwide. The disease can be controlled but not eliminated by using antiviral drugs. Viral resistance and patients' intolerance of the available drugs are growing problems. Consequently, new drugs urgently need to be developed to control the AIDS pandemic. HIV gRNA trafficking along microtubules can form an excellent drug target which the virus cannot easily escape. It can be envisaged that drugs will be developed that disrupt the HIV-microtubule interaction and are not toxic to cells or infected individuals. Before drug discovery can fruitfully be undertaken, a solid understanding of the viral transport processes is however required.