HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

Abstract

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

Fluorescence Microscopy of the HIV-1 Envelope

“…Lipidomic studies provided clear evidence that the lipid composition of the viral envelope differs from that of the plasma membrane of producer cells [2][3][4][5]. The enrichment in sphingomyelin and cholesterol suggests that HIV buds from specialised nanodomains in the plasma membrane, as previously evidenced [6] and further confirmed [7,8]. In particular, cholesterol proved to be a critical lipid, since its depletion from virions inhibits HIV infectivity [9][10][11].…”

“…Recent microscopy studies on live cells confirmed that Gag recruits proteins and lipids at budding platforms [7,8]. Super-resolution experiments (stimulated emission depletion (STED) microscopy in combination with fluorescence correlation spectroscopy (FCS), STED-FCS) in HIV-infected cells proved that fluorescent analogues of cholesterol (labelled with Abberior STAR RED via a PEG-linker) and PIP 2 (PIP(4,5) 2 labelled with ATTO647N) were trapped at Gag assembly sites, as opposed to fluorescent analogues of sphingomyelin (SM, labelled with ATTO647N) and phosphoethanolamine (DPPE, labelled with ATTO647N or Abberior STAR RED) ( Figure 1) [8]. Gag-induced lipid platforms were independently detected at the plasma membrane of transfected cells [7], which were enriched in fluorescent analogues of cholesterol, ganglioside GM1, SM (all TF-labelled) ( Figure 1), and glycophosphatidylinositol (GPI) anchored proteins (labelled with enhanced green fluorescent protein (EGFP)).…”

Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery

“…This system has allowed easy tracking and observation of multiple rounds of ESCRT recruitment in the same VLP. Observing multiple rounds of assembly during HIV budding in T-cells remains technically out of reach, due to the limited membrane and cellular movements of T-cells [58,59].…”

Application of Advanced Light Microscopy to the Study of HIV and Its Interactions with the Host

“…More recently the brightness distribution was used within the cytosol to detect the state of oligomerization of Gag during HIV budding [ 89 ]. A combination of STED and scanning FCS was also used to detect the dynamics of PIP2 during early stages of HIV assembly [ 90 , 91 ]. Fluctuation spectroscopy therefore offers both measurements of dynamics as well as status of molecular aggregates which can be very informative for the study of HIV budding and maturation.…”