How HIV-1 Takes Advantage of PI(4,5)P2 Clusters during Viral Assembly

Abstract

Located at the inner leaflet of the plasma membrane (PM), phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2, here PIP2] has been proposed to exist in distinct pools or microdomains. The origin of such spatially separated pools and the contribution of this distribution to PIP2 function is unknown. PIP2 is critically involved not only in many cellular reactions, but also in HIV-1 particle assembly and release. For example, in vitro at a physiological level of 2 mol%, PIP2 significantly enhances membrane binding of the viral structural protein Gag. And the viral membrane is enriched in PIP2 compared with the PM. Here, we exploited model membranes with PM inner leaflet lipid compositions to characterize PIP2 clustering and to understand how HIV-1 takes advantage of these clusters. Using acyl-chain fluorescently labeled TopFluor-PIP2 in fluorometric assays, we demonstrated that PIP2 begins to form clusters at approximately 0.05 mol%, far below physiological levels. This clustering is dependent on multivalent metal ions. PIP2 clustering behavior is headgroup-specific and independent of acyl chain type. In cells PIP2 is likely to exist in two forms, either as individual PIP2 molecules or else clustered with multivalent metal ions and other PIP2 molecules. Manipulating PIP2 to either form clusters or stay as individual molecules in membranes enabled us to study whether proteins recognize PIP2 clusters, and whether protein binding reorganizes PIP2 distributions. We found that in microscopic-based giant unilamellar vesicle (GUVs) assays, naturally myristoylated HIV-1 MA, the membrane binding domain of Gag, strongly prefers binding to PIP2 in clusters compared with individual PIP2 molecules, whereas charge sensor proteins hardly sense any difference in between individual or clustered PIP2. We are currently examining whether the PIP2 lateral distribution is influenced upon MA binding. We hypothesize that HIV-1 exploits PIP2 clusters as sites of viral assembly.