Quantification of phosphoinositides reveals strong enrichment of PIP₂ in HIV-1 compared to producer cell membranes

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P₂ has been of particular interest since PI(4,5)P₂ is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP₂ in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP₂, but also of PIP₃, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP₂ molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP₂ at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP₂ concentration in this microdomain. These results are consistent with our previous observation that PIP₂ is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.