Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane

Abstract

A critical early event in the HIV type 1 (HIV-1) particle assembly pathway is the targeting of the Gag protein to the site of virus assembly. In many cell types, assembly takes place predominantly at the plasma membrane. Cellular factors that regulate Gag targeting remain undefined. The phosphoinositide phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] controls the plasma membrane localization of a number of cellular proteins. To explore the possibility that this lipid may be involved in Gag targeting and virus particle production, we overexpressed phosphoinositide 5-phosphatase IV, an enzyme that depletes cellular PI(4,5)P2, or overexpressed a constitutively active form of Arf6 (Arf6/Q67L), which induces the formation of PI(4,5)P2-enriched endosomal structures. Both approaches severely reduced virus production. Upon 5-phosphatase IV overexpression, Gag was no longer localized on the plasma membrane but instead was retargeted to late endosomes. Strikingly, in cells expressing Arf6/Q67L, Gag was redirected to the PI(4,5)P2-enriched vesicles and HIV-1 virions budded into these vesicles. These results demonstrate that PI(4,5)P2 plays a key role in Gag targeting to the plasma membrane and thus serves as a cellular determinant of HIV-1 particle production.

Fig. 4.

Gag localizes to PI(4,5)P₂-enriched vesicles induced by Arf6/Q67L. HeLa cells were cotransfected with the HA-tagged Arf6/Q67L expression plasmid and either pNL4-3/KFS/398/PHGFP (A-H) or pNL4-3 (I-L). Arf6/Q67L (blue in A, E, and I) and Gag (red in C and G and green in K) were detected by anti-HA and anti-p17 (MA) Abs, respectively. PHGFP localization is shown in green in B and F. In I-L, cells were also immunostained with anti-CD63 Ab (red in J). Merged images of Gag and PHGFP or CD63 signals are shown in D, H, and L.

Fig. 1.

HIV-1 particle production is inhibited by 5ptaseIV overexpression. (A) HeLa cells were transfected with pNL4-3 alone (-) or were cotransfected with pNL4-3 and expression plasmids encoding 5ptaseIV (wt) or the Δ1 5ptaseIV mutant (Δ1). One day after transfection, cells were metabolically labeled for 2 h with [³⁵S]Met/Cys, and labeled viral proteins in cell and virion lysates were immunoprecipitated with anti-HIV Ig and analyzed by SDS/PAGE followed by fluorography. Viral proteins Pr55^Gag, p24 (CA), and Nef are indicated. (B) Virus release efficiency from pNL4-3-expressing cells was calculated as described in Materials and Methods. Data from three independent experiments were quantified by PhosphorImager analysis and are shown as means ± SD. Note that the reduction in virus release efficiency measured upon 5ptaseIV overexpression may be an underestimate, because a small number of cells in the cotransfected culture express pNL4-3 but not 5ptaseIV (see Fig. 2). (C) HeLa cells transfected with pNL4-3/55FLAG alone (-) or cotransfected with pNL4-3/55FLAG and plasmids encoding 5ptaseIV (wt) or the Δ1 5ptaseIV mutant (Δ1) were analyzed as in A. (D) Virus release efficiency of cells expressing the FLAG-tagged Gag was calculated based on the data from seven independent experiments as described above and in Materials and Methods.

Fig. 2.

Overexpression of 5ptaseIV inhibits Gag localization to the plasma membrane and retargets Gag to a late endosomal compartment. HeLa cells were cotransfected with pNL4-3 and the Myc-tagged 5ptaseIV expression plasmid. Gag (green in A and D) and 5ptaseIV (red in B and blue in C) were detected by mouse monoclonal anti-p17 (MA) and rabbit polyclonal anti-Myc Abs, respectively. The boundary of cells coexpressing Gag and 5ptaseIV is indicated by white dotted lines. Note that, in cells not overexpressing 5ptaseIV (arrow), Gag is localized on the cell surface, whereas, in the 5ptaseIV-overexpressing cells, Gag is predominantly detected in the perinuclear region. In C-F, cells were also immunostained with anti-CD63 Ab (red in E). (F) A merged image of Gag and CD63 signals, with colocalization indicated in yellow, is shown.

Fig. 3.

Expression of the constitutively active Arf6 mutant Arf6/Q67L inhibits virus particle production. HeLa cells were transfected with pNL4-3 alone (-) or were cotransfected with pNL4-3 and an expression plasmid encoding HA-tagged Arf6/Q67L (+) and were analyzed as in Fig. 1. Virus release efficiency was calculated based on data from four independent experiments as described in Materials and Methods. Data are shown as means ± SD.

Fig. 5.

HIV-1 particles assemble in Arf6/Q67L-induced vesicles. HeLa cells transfected with pNL4-3 alone (A and C) or cotransfected with pNL4-3 and the HA-tagged Arf6/Q67L expression plasmid (B and D) were observed by transmission electron microscopy (EM). Higher magnification of boxed areas in A and B is shown in C and D, respectively. Note that virus particles as well as budding structures are detected on the membrane of Arf6/Q67L-induced vesicles (arrows in D). In the cultures cotransfected with pNL4-3 and the Arf6/Q67L expression plasmid, 42% of the virus particles were detected in association with these vesicles, whereas only 6% of virions in cultures transfected with pNL4-3 alone was associated with intracellular vesicles. Scale bars are shown in each panel. Nuclei are indicated (N).