Previous studies have demonstrated that the Gag proteins of several retroviruses recognize PI(4,5)P2
] and that the interaction of Gag MA with PI(4,5)P2
can facilitate protein-protein interactions involved in HIV-1 Gag assembly and trafficking to the site of particle release [2
]. In this study we examined the role of phosphoinositides in EIAV Gag assembly and release. In vitro
, EIAV MA bound several phosphoinositides with different affinities; MA exhibited the highest affinity for PI(3)P. In cells, EIAV Gag accumulated on vesicles enriched in PI(3,5)P2
. By depleting phosphoinositides through over-expression of 5-ptase IV and Sjn-2, we found that the steady-state levels of the PI(4,5)P2
was not critical for Gag assembly, while depletion of phosphoinositides associated with internal membranes interfered with Gag release to a significantly greater extent. Additionally, inhibiting PI(3,5)P2
production from PI(3)P using YM201636 reduced the efficiency of Gag release. Together these observations suggest that targeting to the endocytic compartments containing PI(3)P and PI(3,5)P2
phospholipids is an important aspect of EIAV Gag trafficking and release.
Although HIV-1 is principally found on the plasma membrane, EIAV Gag was found predominantly associated with internal compartments. We showed that a subpopulation of WT EIAV Gag accumulates in endosomes. We speculate that EIAV Gag is normally targeted to endosomal compartments containing PI(3)P and PI(3,5)P2
and sorted from these vesicles back to the plasma membrane by its affinity for PI(4,5)P2
. This notion is supported by observations that VLPs of bothΔp9-Gag, a mutant defective in a very late step in assembly, and K49A-Gag were detected at the cell periphery, as revealed by electron microscopy. The observation that K49A-Gag accumulated in endocytic compartments to ~the same extent as WT Gag () but did not multimerize like the WT in this location () and, moreover, exhibited reduced accumulation on the plasma membrane relative to WT Gag () and was defective for VLP release () suggests that binding the lipids on intracellular vesicles is a critical step. Previous studies demonstrated that PI(4,5)P2
binding triggers major structural reorganization, including inducing the myristyl switch in HIV-1 MA [7
] and changes in HIV-1 and EIAV Gag oligomer formation [11
]. Perhaps EIAV Gag interaction with PI(3)P and/or PI(3,5)P2
in particular induces conformational changes that facilitate productive EIAV Gag multimerization. After all, EIAV MA binds these phospholipids with high affinity while HIV-1 Gag does not bind them at all (7, 11). Presumably, changes resulting from mutation of S100 alter the route, but do not prevent, trafficking through these endocytic compartments. Indeed, compared to the WT Gag protein, relatively more S100A accumulated on the cytoplasmic face of PI(3)P- and PI(3,5)P2
-containing compartments ().
An alternative to the notion that PI binding induces conformational changes required for productive assembly and release is the idea that, in contrast to HIV, PI binding is NOT required for EIAV Gag trafficking. In this case, the defect in viral particle release following mutation of K49 would be attributable to its potential role in trimer stabilization or formation of higher-order assemblages of the trimer (c.f., ). We do not favor this interpretation (i) because EIAV and HIV MA exhibit structural and functional conservation and, although EIAV lacks N-terminal fatty acid modification, we expect that it conserves the PI-induced conformational change that occurs with HIV. Moreover (ii), although mutation of K49 to Ala did not alter Gag localization as revealed by confocal microscopy, it exhibited striking defects when examined by electron microscopy (c.f., ). Similar mutation of S100, a PI-binding pocket residue that is distal to K49 in the MA structure and not part of the trimer interface resulted in similar budding defects. Most likely, the mutations interfered with interactions between the lipid-interacting Gag protein assemblages and PI(4,5)P2-membrane microdomains at the budding site. S100A was released more efficiently than K49A; perhaps it is blocked at a later budding stage: We noted that its membrane tether appeared to be thinner. (iii) In the BiFC assay (c.f., ), K49A exhibited reduced Gag-Gag interaction in the cell interior but not at the periphery. If its release defect was exclusively due to disruptions in trimer stabilization or formation of higher-order assemblages of the trimer, defective protein-protein interaction should have been detected throughout the cell.
Further investigations to define the precise role of phosphoinositides in retroviral assembly, trafficking and release are warranted and may reveal new opportunities for development of antiviral strategies.