PIP₂ promotes the incorporation of CD43, PSGL-1, and CD44 into nascent HIV-1 particles

Determinants regulating sorting of host transmembrane proteins at sites of enveloped virus assembly on the plasma membrane (PM) remain poorly understood. Here, we demonstrate that the PM acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates this sorting into an enveloped virus, HIV-1. Incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles has profound effects on viral spread; however, the mechanisms promoting their incorporation were unknown. We found that depletion of cellular PIP₂ blocks incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles. Expansion microscopy revealed that PIP₂ depletion diminishes nanoscale coclustering between viral structural protein Gag and the three transmembrane proteins at the PM and that Gag induces PIP₂ enrichment at its vicinity. CD43, PSGL-1, and CD44 also increased local PIP₂ density, revealing their PIP₂ affinity. Together, these results support a previously unknown mechanism where local enrichment of an acidic phospholipid drives coclustering between viral structural and cellular transmembrane proteins, thereby modulating the content, and hence the fate, of progeny virus particles.