Abstract
HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4, 5) bisphosphate, 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 was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T 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 show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.
Introduction
During HIV-1 replication cycle, the assembly and release of newly made viral particles occurs at the plasma membrane of the infected cells (Fig 1a). Budded HIV-1 particles display an unusual lipid membrane composition that differs significantly from that of the plasma membrane of the host cell. It is enriched in sphingomyelins (SPMs), glycosphingolipids, cholesterol (Chol), and phosphoinositides, such as phosphatidylinositol (4, 5) bisphosphate lipid (PI(4,5)P2)(1, 2) and characterized by highly ordered, i.e. densely packed, lipid organization (1, 3). This raises the question on how virus components interact with lipids in the plasma membrane of the host cells to gather such specific lipid environment during assembly. Previous studies of HIV-lipid interactions were mainly based on in vitro model systems (4–8), tagged phospholipid interacting protein domains (9), or virus lipidomics (1, 2, 4). However, direct observations of the relevant molecular interactions in the living cells have for long been challenged by the limited spatial resolution of conventional optical microscopy. Fortunately, recent advances in super-resolution microscopy techniques have enabled for characterization of molecular mobility at the nanoscale, i.e. on the scale of individual (< 140 nm) HIV-1 particles. For example, experiments employing a combination of super-resolution STED microscopy and fluorescence correlation spectroscopy (STED-FCS (10) or scanning STED-FCS, sSTED-FCS (11, 12)) revealed a low mobility of molecules on HIV-1 surface (3) due to the high degree of lipid packing in the virus membrane (12, 13). The retroviral protein Gag is thought to be a key player in altering the HIV-1 lipid environment as it is not only the main structural determinant of the particle assembly, but it also mediates interactions between assembling virus particle and plasma membrane lipids. HIV-1 Gag is composed of 4 domains, MA, CA, NC and p6 and 2 spacer peptides (sp1 and sp2). The MA domain of Gag is myristoylated on its N-terminus and contains a highly basic region (named HBR), both responsible for Gag anchoring and targeting to the cell plasma membrane (PM)(14). The CA domain is involved in Gag-Gag interaction for multimerization. The NC domain is required for genomic RNA encapsidation and also for Gag-Gag multimerization through RNA (15). The p6, and part of the NC domain, recruit ESCRT cell factors for particle budding (16). The space peptide sp1 is also required for proper particle assembly and budding (17). The binding of Gag to the plasma membrane is primarily resulting from a bipartite signal within MA (reviewed in (14)). It is understood that MA is thought to interact with the acidic lipids of the PM inner leaflet(18), although recent studies suggest that the NC domain of Gag could also play a role(19). Substitutions of the MA basic residues have shown to affect targeting and membrane binding of HIV-1 Gag both in liposomes (20) and in cells (21). Furthermore, a lipidomic study has suggested that Gag interacts with acidic phospholipids in cells since virions produced by HIV-1 infected cells were enriched in PI(4,5)P2 via a MA dependent manner (2), and this interpretation is supported by recent report using tunable PI(4,5)P2 level to track Gag anchoring onto cell membrane via imaging analysis (22). The unusual lipid contents in HIV show that HIV particles require a specific lipid membrane environment for HIV-1 assembly. A key question in HIV assembly is whether Gag is targeted toward pre-existing lipid domains at the plasma membrane, or whether Gag actively creates its unique lipid compositions for virus formation. Historically, Gag has been shown to associate with the detergent resistant membrane (DRM) fractions (23, 24), which are enriched in SPM and Chol, and rafts properties of virion-cholesterol impact on virus function (25, 26). Saad et al.(27) have used truncated acyl chains PI(4,5)P2 to show the unsaturated sn2 acyl chain of the PI(4,5)P2 is trapped in an hydrophobic pocket while the myristate embed into the lipid membrane, leaving the complex MA:PI(4,5)P2 to partition to more liquid ordered membrane environments. Using phase separated giant unilamellar vesicles (GUV), Keller et al., have shown that multimerizable MA are partitioning into more liquid disordered membrane environments (6). In addition, using coarse grained molecular dynamics, researchers have highlighted that HIV-1 MA interacts only with PI(4,5)P2 sugar head and that PI(4,5)P2 is concentrated within the immediate vicinity of MA (28). Recent NMR studies on HIV-1 MA in interactions with lipidic membranes confirmed these results (29). Finally, our recent lipidic membrane-based study demonstrated that recombinant HIV-1 Gag was able to segregate PI(4,5)P2, and cholesterol, but excluded SPM upon Gag multimerization, and that Gag preferred to partition into PI(4,5)P2 enriched liquid disordered lipid membrane environments on GUV rather than liquid ordered lipid membrane environments (7). Together, these results suggested that HIV1 Gag may generate its own PI(4,5)P2 enriched membrane environment at the cell plasma membrane rather than assembling on pre-existing areas or domains with distinct lipid membrane environments. This is consistent with a study utilizing a novel biological tool to tune PI(4,5)P2 level at the cell plasma membrane, recently reported that PI(4,5)P2 not only targets Gag to the cell membrane but it is necessary for assembly site formation (22). In order to study this dynamic equilibrium of Gag-lipid interaction in living host CD4+ T cells, here we employ super-resolution sSTED-FCS approach to analyse lipid mobility inside and outside HIV-1 assembly sites on fluorescent lipid labelled CD4 T cells infected by HIV-1 or expressing Gag only. We find that infectious HIV1 is able to immobilize PI(4,5)P2, and Chol, but not phosphatidylethanolamine (PE) and SPM in these host cells, and that only Gag is required to restrict the movement of these lipid molecules at the assembly sites. Our results highlight that HIV-1 Gag selectively trap PI(4,5)P2 and Cholesterol in host CD4 T cells to create its own specialized lipid membrane environment for virus assembly.
Results
PI(4,5)P2 and Cholesterol, but not sphingomyelin, are trapped during HIV-1 assembly in infected CD4+ T cells
Assembly of fully infectious HIV-1 particles requires a concomitant assembly of viral Env and Gag/GagPol at the assembly site, i.e. at the cellular plasma membrane, involving specific lipid environments that can be either pre-existing or generated by the viral proteins. To distinguish these possibilities, we infected Jurkat CD4+ T lymphocytes with infectious NL4.3 HIV-1 viruses. These viruses expressed green fluorescent protein (GFP), which was inserted between MA and CA domains of Gag (Gag-iGFP) (30). The concomitant fluorescence allowed to track the movement of fluorescently tagged lipids at HIV-1 assembly sites (Fig. 1a). 72 hours post infection we tested whether infected cells are able to support HIV1 assembly. Time lapse imaging at 37 ° C of cells adhered to poly-L-Lysine coated coverslips highlighted that infected cells produced new virus assembly sites within 20 minutes post adherence (Fig 1b) This is consistent with previous HIV1 assembly studies of HIV-1 particles (31) and HIV-1 Gag virus-like-particles production in CD4+ T cells (32). To visualize lipids mobility within or outside of virus assembly sites, we labelled the CD4+ T cells with fluorescent lipid analogues prior to adhering cells to poly-L-Lysine coated coverslips. The selected analogues correspond to lipids known to be enriched in HIV-1 membrane such as Chol and SPM, to have major role in Gag membrane-interaction such as PI(4,5)P2, or to be selectively excluded in HIV membrane such as phosphatidylethanolamine (PE). To acquire lipid mobility data via sSTED-FCS, HIV-1 assembly sites were aligned with the laser scanning orbit of our microscope, using Gag-iGFP fluorescence signal as a guide (Fig 1c). For this we first took larger confocal microscope images identifying spots of GFP fluorescence as relevant Gag clustering sites. For our analysis, we selected only small and immobile Gag clusters at the T cell surface with low fluorescence Gag signal intensity. Previous studies have shown that such sites represent virus assembly that is still in progress (32), and thus minimize the risk of performing lipid mobility analysis on the site of already budded virus particles. After this identification step, the microscope was switched from imaging (x-y axis) to continuous orbital line scanning (x-t axis, intensity carpet acquisition) mode. Consequently, we recorded temporal fluorescence intensity fluctuations at each position (or pixel) along the scanned orbit. An orbit always included Gag-clustering sites and thus HIV-1 assembly sites (GFP fluorescence) and areas without Gag (no GFP fluorescence) (Fig 1). We firstly acquired a short intensity carpet for Gag fluorescence in confocal mode to ensure that a) the scanning orbit was correctly overlapped with the position of the virus assembly site; and b) the Gag assembly site was sufficiently immobile to enable lipid mobility measurements (Fig 1d). Afterwards, we acquired an intensity carpet for the red-emission signal of the fluorescent lipid analogues with a reduced observation spot size (diameter 100 nm, STED microscopy mode) (Fig 1e). The reduced observation spot size ensures that we are indeed mainly covering lipid data at the assembly site only, and not averaging over a larger non-relevant area. Finally, we again recorded a fluorescence intensity carpet of Gag fluorescence in confocal mode to further ensure that the virus assembly site shows no detectable drift during the whole acquisition process (Fig 1f). The acquisition parameters for the recording of the fluorescent lipid analogue data were chosen for reducing bias and for generating accurate data: 1) The spot size of 100 nm in diameter ensured sufficient spatial resolution for distinguishing trapped from non-trapped lipids (see results further on) and yet still high enough signal-to-noise ratios for reasonable and reproducible FCS data (see error bars further on). 2) Orbital beam-scanning with 3.23 kHz realized a low enough dwelling at each scanned point to minimize (excitation and STED) laser exposure and thus phototoxic effects as well as a high enough temporal resolution of the decaying FCS data (34). 3) The number of total points (30) per orbital scan facilitated the collection of sufficiently enough data points for an accurate identification of the virus assembly sites (Fig. S1) (11). FCS data were generated for each point along the orbital scan and fitted with a generic twodimensional (2D) diffusion model to obtain the average transit times through the observation spot and the diffusion coefficient of the lipid analogues at each location (Fig 1g). Figure 2 shows the change in lipid lateral mobility observed in infected Jurkat T cells within (red boxes) and outside of (green boxes) the HIV-1 assembly sites. The lateral mobility of the lipids as measured in uninfected control cells are presented in blue. As previously observed for PI(4,5)P2 in SLBs ((7) and Fig. S2), the median diffusion coefficient of PI(4,5)P2 (Fig 2a) within the assembly sites was strongly reduced (D = 0.02 µm2/s) compared to the one observed outside of the assembly sites (D = 0.17 µm2/s) or in HIV negative cells (D = 0.20 µm2/s). Therefore, in infected Jurkat T cells, general mobility of PI(4,5)P2 was 8to 10-fold decreased within the assembly sites compared to elsewhere or in HIV negative cells. We further tested Gag specific mobility of Chol. Chol has been shown to play a major role in the assembly process of HIV-1 virus(23). When monitoring the diffusion coefficient of Chol (Fig 2b), we observed a similar decrease of Chol mobility (D = 0.10 µm2/s) within the assembly sites compared to elsewhere (D = 0.25 µm2/s) and in HIV negative cells (D = 0.29 µm2/s). In contrast, SPM did not exhibit any significant change in mobility in the presence or absence of HIV-1 or within and outside the assembly sites (Fig 2c; D = 0.15 and 0.17 µm2/s within and outside the assembly sites, respectively, and D = 0.15 µm2/s in uninfected cells) Similarly, we observed no significant change in the mobility of the PE lipid analogue within (D = 0.32 µm2/s) versus outside (D = 0.31 µm2/s) the assembly sites or versus uninfected cells (D = 0.31 µm2/s) (Fig 2d).
Expression of HIV-1 Gag alone is sufficient to immobilize PI(4,5)P2 and Cholesterol during formation of Virus Like Particles
HIV-1 Gag alone is able to self-assemble at the plasma membrane of the Jurkat T cells, leading to the generation of immature virus-like-particle (VLP). Kerviel et al. (35) proposed that self-association of Gag is sufficient to generate a specific lipid environment. Therefore, by transfecting HIV-1 Gag only expression vector into Jurkat T cells and measuring changes in the mobility of lipids at Gag self-association sites, we tested whether the selective lipid clustering we have observed in HIV-1 infected cells (see above) were also present in Gag only assembly sites. As with the infected Jurkat T cells, we have firstly tested for the evidence of appearance of VLP assembly sites at the surface of HIV Gag expressing Jurkat cells (Fig 3a), followed by the determination of lipid diffusion coefficients both within and outside of those sites. We determined lipid diffusion at arbitrary sites in non-transfected (non-Gag expressing) cells as control. Figure 3b-e depicts the different values of diffusion coefficients determined within (red boxes) and outside of the assembly sites of HIV Gag VLP (green boxes), and in the non-transfected control cells (blue box). As observed in infected cells, the lateral mobility of PI(4,5)P2 was significantly reduced inside (D = 0.11 µm2/s) compared to outside of assembly sites or to non-transfected cells (D = 0.24 µm2/s and 0.20 µm2/s, respectively) (Fig 3b). However, the reduction in mobility between inside and outside the assembly sites is only 2.2-fold (0.24/0.11) for HIV Gag VLP only expressing T cells in comparison with 8.5-fold (0.17/0.02) for HIV infected T cells. Nevertheless, experiments on model membranes support the direct role of Gag in this process of PI(4,5)P2 trapping. Upon addition of Gag to supported lipid bilayers (SLBs) with 0.2% of fluorescent PI(4,5)P2 we observed the formation of PI(4,5)P2 clusters at the Gag binding sites with a concomitant 7-fold decrease in PI(4,5)P2 mobility at these sites compared to elsewhere on the SLBs or on SLBs in the absence of Gag (Fig. S2). Also in line with our data on infected T cells, we also observed a 2-fold reduction in the lateral mobility for Chol within the Gag assembly sites (D = 0.16 µm2/s) of HIV Gag VLP only expressing T cells, compared to either outside of assembly site (D = 0.30 µm2/s) or in non-transfected control cells (D = 0.29 µm2/s, Fig 3c). In contrast, SPM and PE again exhibited no significant changes in their lateral mobility between HIV-1 Gag self-assembly sites when compared to outside the sites or in non-transfected cells (Fig 3d and Fig 3e respectively).
HIV-1 Gag is the primary determinant of lipid trapping efficiency at HIV-1 assembly sites in CD4+ T cells
The decrease in lateral mobility of PI(4,5)P2 and Chol at the Gag assembly sites suggests trapping of these lipids during virus particle formation. To quantify this restricted diffusion further, we utilized a confinement index based on normalized relative cumulative frequency histograms of the values of the diffusion coefficient D (for D = 0.001 to 1 µm2/s, normalized to 100 for D = 1 µm2/s). Representative cumulative frequency histograms for diffusion within and outside of the assembly sites are shown in Figure 4a for the fluorescent PI(4,5)P2 lipid analogue (and in Fig. S3, S4 for other lipid analogues), highlighting a huge difference for PI(4,5)P2 and Chol (but not for SPM and PE). To specify this difference further, we classified three diffusion regimes, “fast”, “intermediate”, and “slow” mobility, for respective values of D ≤ 0.01 µm2/s, 0.01 D ≥ 0.1 µm2/s, and D ≥ 0.1 µm2/s, we calculated the sum of cumulative frequencies within each regime, and plotted the confinement index for each regime as ratio of the latter values for lipid diffusion within and outside of the assembly sites, or within the assembly sites and non-infected (or non-transfected) cells. For an increased confinement of lipids inside the assembly sites, we would expect a larger relative fraction of low values of D, i.e. an increased fraction and thus confinement indices > 1 within the “intermediate” and especially the “slow” mobility regime (and consequently a reduced fraction and thus a confinement index < 1 within the “fast” regime). In contrast, similar cumulative frequency histograms and thus confinement indices 1 for all regimes indicate no change in mobility and thus no confinement within the assembly sites. As expected, the various values of confinement indices confirm our previous conclusion, i.e. that SPM and PE did not exhibit any confinement within assembly sites, while PI(4,5)P2 and Chol revealed a drastic reduction of lateral mobility and thus confinement at the assembly sites. When we compared the confinement index values obtained for the different lipids in HIV-1 Gag transfected cells (Fig 4b, grey bars) against those in HIV-1 infected cells (Fig 4b, black bars), we observed similarities between these two conditions. Specifically, both PI(4,5)P2 and Chol have values of high confinement indices > 1 in the “intermediated” and “slow” regime for both HIV infected and Gag-only expressing T cells, while values 1 of confinement indices are conserved for both SPM and PE. This highlights that the Gag protein on its own seems to be sufficient to facilitate the confinement or trapping of cholesterol and PI(4,5)P2 in HIV assembly sites, and no other molecular factor only present in infected cells (and not in the Gag-only case) is required.
Discussion
How enveloped viruses acquire their lipid membrane shell is of great importance for their biogenesis and their capacity to infect cells. In the case of HIV-1, recently it has been shown that the envelope lipid composition was a determinant of low molecular mobility of the Env protein (3, 13). Several works have been conducted to reveal different envelope viruses having their own unique lipid composition (2, 4, 6, 36), implying their distinct biological requirements during infection, and suggesting their capacity in sorting specific host lipids into their virus membrane. Two main lipids and a sterol have been described to play a major role for HIV-1 biogenesis and infectivity. PI(4,5)P2 is known to be the plasma membrane lipid targeted by the MA domain of the polyprotein Gag (20, 21, 27, 37). Sphingomyelin and Cholesterol enriched lipid environments, often denoted “raft” like domains, have also been described as essential for envelop retrieving (38), HIV-1 assembly (23, 39, 40) or infectivity (24, 25). However, it is unknown whether a pre-existing cellular lipid complex or a virus induced lipid platform is the source of virion lipid membrane during HIV-1 formation (35). Lipid sorting and lipid domain generation in viral assembly can be identified by nanoscale spatial changes in lipid mobility at the plasma membrane (10, 11), and our super-resolution microscopy based approaches for the analysis of the lipid dynamics in living cells represent the most relevant model of observation. In this study, we have determined the diffusion coefficients of lipids by applying scanning STED-FCS (sSTED-FCS) on HIV infected, Gag expressing, and HIV negative CD4+ Jurkat T cells. sSTED-FCS approach allows for direct simultaneously quantitative comparison of lipids mobility both within or outside assembly sites at the plasma membrane. In comparison with the plasma membrane of HIV-1 negative cells or plasma membrane regions outside of HIV-1 assembly site, the mobility of PI(4,5)P2 and Chol within viral assembly sites is drastically reduced (up to 10 times decrease of median D). Interestingly, the same trend was observed on the assembly site of Gag VLPs in cells expressing only Gag. Indeed, although to a less extent than in HIV-1 producing cells, the median values of the diffusion coefficients of PI(4,5)P2 and Chol were significantly reduced in the VLP assembling area. In contrast, the mobility of neither SPM nor neutral lipid PE exhibited any variation in median values, for both HIV-1 producing or Gag VLPs producing cells. It has been recently shown that diffusion coefficient distributions could unravel the nature of lipid motions (41). Therefore, to allow direct comparison of the virus component effect on lipid mobility, independently of the cell or the number of expressed component (Gag alone or infectious virus), a confinement index was defined. As in more detail described within the result section, it is based on the relative changes in three different parts of the normalized cumulative distribution of the logarithm of the diffusion coefficients observed inside and outside the virus or VLP assembling site. The value of this confinement index into these three parts reflects the relative increase in lipids that are trapped (“slow”, D < 0.01 µm2/s), confined (“intermediate”, 0.01 < D < 0.1 µm2/s) or freely diffusing (“fast”, D > 0.1 µm2/s) at the assembly sites. By definition, the confinement index will be equal to 1 if no effect occurs on lipid mobility inside the assembly when compared to outside the assembly site. This unit value was always observed with the PE lipid which is generally used as a control of free diffusing lipid in cell plasma membranes (10) and is not described as being enriched in HIV-1 envelop (1, 4). Amongst the different lipids tested here, the PI(4,5)P2 exhibited the strongest confinement index for the slow part of the distribution, (c.i. 10) both for HIV-1 virus and VLP assembly sites in CD4+ T cells. This reveals that transient binding of PI(4,5)P2 to aggregating Gag conjugated PI(4,5)P2 into the nascent virion or the VLP. This assembly site specific trapping of PI(4,5)P2 seems to be indeed exclusive to Gag since the observed confinement indices showed the same trend independent of assembling HIV-1 or VLPs. Moreover, similar confinement indices were observed on the SLB in the in vitro system tested here, i.e. SLBs made of PC, PS and fluorescent PI(4,5)P2 with or without addition of self-assembling HIV-1 Gag protein (see Supplementary Data 2). Similar to PI(4,5)P2, the confinement index of slow diffusing Chol was also above 1 in infected cells as well as in transfected cells, confirming that Chol is trapped during HIV-1 assembly and that Gag alone is sufficient to restrict cholesterol mobility at the assembly site. Consequently, restriction of PI(4,5)P2 and Chol seems to be independent of non-Gag viral proteins, such as HIV-1 Env or accessory/regulatory proteins. Using in vitro lipid membrane models of different lipid compositions, we recently reported (7) that Gag was able to generate PI(4,5)P2-cholesterol but not SPM enriched lipid nanodomains. This is consistent with data observed here in HIV1 producing or Gag expressing CD4+ T cells. Notably, such local enrichment of PI(4,5)P2 and Chol lipids at virus assembly sites has been predicted using coarse grain molecular dynamics in the case of the matrix self-assembly of Ebola virus (42), implying a potential general mechanism for a number of enveloped viruses that bud from the host cell plasma membrane. Enrichment of specific lipid in HIV-1 viruses has already been observed (2, 4, 23) but the mechanism remains unknown. Different values of virion lipid enrichment have been found, depending on the cell lines and the biochemical methods to purify viruses or isolate plasma membranes from the other cell membranes (1, 2, 4). In H9 T lymphocytes, Chan et al. have shown that HIV-1 lipid envelop is two times higher in PI(4,5)P2 and three times higher in cholesterol when compared to the plasma membrane of the HIV-1 producing H9 cells. Our results show that this enrichment occurs during virus assembly. Given that PI(4,5)P2 is important for the binding of Gag to the cell plasma membrane (28), the clustering of PI(4,5)P2 at the cell membrane may provide an additional mechanism to further concentrate Gag thereby enhancing virus assembly. Furthermore, molecular simulations of PI(4,5)P2 clustering has revealed the induction of negative membrane curvature (43) by PI(4,5)P2, which could facilitate the formation of spherical particle structure during virus assembly. Cholesterol has been shown to play a role in HIV-1 Gag matrix binding to membrane(5, 44) as well as in the stabilization of PI(4,5)P2 lipid nanodomains in the absence of proteins (43). Therefore, it would be difficult to discriminate whether cholesterol trapping is a consequence of PI(4,5)P2 membrane enrichment at HIV-1 assembly sites or is due to a direct interaction with Gag. Nevertheless, the cholesterol confinement index found in this work is much lower than the one observed for PI(4,5)P2, allowing the conclusion that the incorporation of cholesterol at the HIV-1 assembly site is more of a consequence of PI(4,5)P2 enriched membrane environment than a strong direct interaction with Gag. It has been suggested for a long time that pre-existing sphingomyelin-cholesterol enriched cell plasma membrane nanodomains could play a role in HIV-1 assembly (reviewed in (38)). Therefore, changes in SPM dynamics at the virus assembly site were also assessed using our sSTED-FCS approach. The observed confinement indices of SPM were never above 1, highlighting that HIV-1 assembly has no effect on the SPM mobility at the CD4+ T cell plasma membrane. Overall our results provide direct evidence that HIV1 Gag assembly drives the lipid sorting in host CD4 T cell plasma membrane and creates its own PI(4,5)P2/cholesterol lipid enriched membrane environment likely to provide a positive feedback loop to recruit additional Gag proteins at the budding site through PI(4,5)P2-Gag interactions for the biogenesis of HIV-1 particles.
Material and Methods
Experimental Design
The main goal of this study was to unravel whether the viral Gag protein is targeted towards preexisting lipid domains at the host cell plasma membrane that will help in HIV-1 assembly, or whether, in the opposite, Gag creates its own lipid domain during virus formation. In order to do so, we measured the change in mobility of different lipids at HIV-1 assembly sites. We used lipids known to play a role either in Gag membrane targeting (PI(4,5)P2) and assembly (Cholesterol) or known to be part of pre-existing lipid domains at the plasma membrane (SM) and finally a control lipid (PE) not described to play a role in any of the two possible mechanisms. To achieve our goal, our experimental setup was based on a direct measurement of apparent diffusion coefficient of the lipids inside and outside HIV-1 Gag assembly sites using spatial scanning fluorescence correlation spectroscopy on a super-resolution STED microscope (delivering an observation spot size of around 100 nm in diameter, i.e. below that of a conventional microscope). Our measurements were performed in HIV-1 infected CD4 T-cells and as a control in CD4 T-cells expressing Gag only. Finally, using a top-bottom approach of purified HIV-1 Gag on PI(4,5)P2supported lipid bilayers we could highlight that Gag was sufficient to generate a PI(4,5)P2-nanodomain platform for assembly.
Lipids and fluorescent lipid analogues
Lipids 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC), brain L-αphosphatidylserine (PS) and brain Phosphatidylinositol 4,5biphosphate (PI(4,5)P2) were purchased from Avanti Polar lipids (Alabaster, AL, USA). ATTO 647N – 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine (ATTO647N-DPPE) and ATTO 647N – Sphingomyelin (ATTO647N-SM) were purchased from Atto-Tec GmbH (Siegen-Weidenau, Siegen, Germany). Abberior STAR RED (KK114) 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (KK114-DPPE), Cholesterol – PEG(1k) Abberior STAR RED (KK114) (Chol-PEG(1k)-KK114), and ATTO 647N phosphatidylinositol 4,5-biphosphate (ATTO647NPI(4,5)P2) were all purchased from Abberior GmbH (Göttingen, Germany).
Gag protein and Supported Lipid Bilayers
Full length Gag protein has been produced and purified as described previously(45). Supported Lipid Bilayers (SLBs) for Gag lipid clustering measurements were prepared from 30nm small unilamellar vesicles (SUVs) with DOPC - 68%, PS - 30%, PI(4,5)P2 - 1.9% and ATTO647N-PI(4,5)P2 0.1% mol at 0.1 mg/mL in a citrate buffer (Na Citrate 10mM, 100 mM NaCl, and 0.5 mM EGTA, pH 4.6). The SUV were then spread for 45 min at 37°C on coverslips pre-treated by sonication in 5% SDS solution and incubation for 30 min in freshly made pi-ranha solution (H2SO4:H202 2:1 vol). After disposition of the SLBs scanning FCS measurements were performed and again 15 minutes after Gag was added at 1µM final concentration.
Plasmids
Plasmid expressing fully infectious T cell tropic NL4.3 HIV-1 Gag-iGFP was previously described (30) and was a kind gift from Dr Benjamin Chen. Plasmid expressing NL4.3 HIV 1 Gag eGFP fusion protein was previously described (45) and was a kind gift from Dr Hugues de Rocquigny.
Cell culture
Jurkat T cells (Human T cell leukemia cell line– ATCC TIB-152) were grown in RPMI-1640 with Glutamax (GIBCO), supplemented with 10 % foetal calf serum and, 100 U/mL penicillin-streptomycin and 20mM HEPES pH 7.4. Cells were maintained at 37 °C, 5 % CO2.
NL4.3 HIV-1 Gag.iGFP particle preparation
Fully infectious NL4.3 HIV-1 Gag-iGFP particles were prepared from the tissue culture supernatant of 293T cells transfected using polyethyleneimine (PEI) with 15 µg pNL4.3 HIV-1 GagiGFP plasmid. Tissue culture supernatants were harvested 48 h after transfection, and particles were concentrated using Lenti-X Concentrator reagent (Clontech) according to the manufacturer’s instructions. Concentrated particles were snap frozen and stored in aliquots at −80 ° C
Jurkat T cell infection
Jurkat CD4+ T cells were infected by incubation of 1 million cells with HIV-1 NL4.3 Gag-iGFP particles in 50 uL RPMI medium for 1 h at 37 °C. Cells were washed three times in RPMI medium and then cultured for 72 h at 2 million cells/mL to achieve 5-10 % infection rate with progeny virus production.
Jurkat T cell transfection
Jurkat T cells (2×106) were microporated with 4 µg of plasmid expressing NL4.3 HIV-1 Gag eGFP, using the AMAXA system (Lonza), as previously described (46). After microporation, Jurkat T cells were incubated in RPMI complete medium, and harvested 24 h posttransfection for microscopy acquisitions as described in the following sections.
Microscope setup
Scanning STED-FCS measurements were performed on the Abberior Instrument Expert Line STED super-resolution microscope (Abberior Instruments GmbH, Göttingen, Germany) using 485 nm and 640 nm pulsed excitation laser sources and a pulsed STED laser operating at 775 nm and 80 MHz repetition rate. The fluorescence excitation and collection was performed using a 100×/1.40 NA UPlanSApo oil immersion objective (Olympus Industrial, Southend-on-Sea, UK). All acquisition operations were controlled by Imspector software (Abberior Instruments) and point FCS data for the calibration of the observation spot sizes was recorded using a hardware correlator (Flex02-08D, correlator.com, operated by the company’s software). Confocal microscope FCS measurements were performed on a Zeiss LSM 780 (Zeiss, Iena, Germany) using a HeNe 633 nm laser as the excitation source. Fluorescence excitation and collection was performed using a 63×/1.40NA PlanApo oil immersion objective (Zeiss). Acquisition operations were controlled by the Zeiss Zen software. Point FCS was used to calibrate the observation spot with the LSM 780 internal hardware correlator.
Microscope sample preparation
For calibration of the observation spot sizes using STED-FCS, supported lipid bilayers (SLBs) were prepared, as previously described (47), by spin coating a coverslip with a solution of 1 mg/mL DOPC and 0.5 μg/mL KK114-DPPE in CHCl3/MeOH. Coverslips were cleaned by piranha solution (3:1 sulfuric acid and hydrogen peroxide). The lipid bilayer was formed by rehydrating with SLB buffer containing 10 mM HEPES and 150 mM NaCl pH 7.4. Calibration of the confocal observation spot size was made with a 50 nM solution of Rhodamine in pure water using D=280µm2.s-1 as the diffusion coefficient. For lipid mobility measurements inside and outside of Gag assembly sites in mammalian cells 3×105 Jurkat T cells 72 h post-infection with NL4.3 HIV-1 Gag-iGFP virus particles or 24 h post-transfection with NL4.3 HIV-1 Gag-eGFP expressing plasmid were resuspended in 200 µL of Leibovitz’s L 15 medium (Sigma) and incubated with 25 nM of the indicated fluorescent lipid analogue probe for 5 min at 37 °C. Cells were then washed three times in L-15 medium, resuspended in 250 µL L-15 medium and adhered to poly-Llysine (Sigma) coated glass surface of 8-well Ibidi µ-Slides (Ibidi, Martinsried, Germany) at 37 °C prior to microscopy measurements. For lipid mobility measurements on Myr(-) Gag containing SLBs, prior to experiments citrate buffer was changed to protein buffer (Hepes 10 mM, pH=7.4, KCl 150 mM, EDTA 2mM), followed by injection of 1 µM Myr(-)Gag.
Scanning (STED)-FCS signal acquisition
sSTED-FCS data of the lipid mobility inside and outside Gag assembly sites in Jurkat T cells was acquired at 37 °C using fluorescent Gag signal as a guide. Fluorescent lipid signal intensity fluctuation carpets (scanning an orbit multiple times) were recorded using the Imspector software with following parameters: orbital scan frequency 3.23 kHz, scan orbit length 1.5 µm, pixel dwell time 10 µs, total measurement time 10 s, pixel size 50 nm/pixel, 10 µW excitation power (back aperture) at 640 nm, observation spot diameter 100 nm FWHM (as determined by SLB calibration measurements – Supplementary Data 1). The scan frequency selected for these experiments was set sufficiently high in order to enable the extraction of the diffusion dynamics of fluorescent lipid analogues in the cell plasma membrane (11).
FCS curve autocorrelation and fitting
The FoCuS-scan software(34) was used to autocorrelate the scanning STEDFCS data, and to fit it with a classical 2D diffusion model: where GN(τ) is the correlation function at time lag τ GN(∞) the offset, GN(0) the amplitude, txy the average lateral transit time through the observation spot. As done in all previous scanning STED-FCS measurements, we left outanomaly factors in the fitting equation (as often done in non-scanning single point STED-FCS data), since the decay of the scanning STED-FCS curves starts at 0.5-1 ms correlation times only and because anomaly is much better revealed by comparing (cumulative) frequency histograms of resulting parameters(11). Values of the molecular diffusion coefficient (D) were calculated from txy: where FWHM represents the usual full-width half maximum of the observation spot (100 nm, see Supplementary Data 1).
Calculation of the confinement index
Diffusion coefficients obtained for the different lipid in the different conditions tested were binned in groups every 10−3µm2/s from 0 to 1.2 µm2/s. The distribution was cumulated and normalized. It was then split into three parts: Slow (D < 10−2µm2/s), intermediate (10−2 ≤ D < 10-1 µm2/s) and fast (D ≥ 10−1µm2/s). The relative proportion (Pi) of the total distribution (0 ≤ Pi ≤ 1) of each of these parts were then calculated for each molecule inside assembling site, outside assembling site and in non-infected nor transfected cells. The confinement index is defined for each of the three parts as the quotient of the Pi value found inside the assembly site over the Pi value found outside the assembly site or in non-infected nor transfected cells. (Fig 4).
Statistical analysis
Due to the non-Gaussian nature of the diffusion coefficient data distribution, Wilcoxon test was used with the result p < 0.05 considered statistically significant. Statistical tests were performed using Graphpad Prism software. Power calculations confirmed that for chosen sample sizes the power of a two-sided hypothesis test at the 0.05 significance was over 90%. In all compared groups interquartile range (IQR) serves as an estimate of variation. IQRs were similar in all compared groups.
ACKNOWLEDGEMENTS
We would like to thank Thomas J Hope (Northwestern University, Chicago) and the Department of NanoBiophotonics (Max Planck Institute for Biophysical Chemistry, Gottingen, Germany) for the eGFP.Vpr construct. We would also like to thank the Montpellier RIO Imaging facilities (MRI, Montpellier France) and the Wolfson Imaging Centre Oxford (UK), for help with the use of microscopes. Cyril Favard and Delphine Muriaux are members of the CNRS consortium ImaBIO, France. The overall project was funded by CNRS and the French National Agency for Research (ANR “FLUOBUDS”, ANR-13-BSV5-0006-01) and the French Agency against AIDS (ANRS). JC and CE were supported by the Medical Research Council (grant number MC-UU-12010/unit programs G0902418 and MCUU-12025), MRC/BBSRC/EPSRC (grant MR/K01577X/1), Wellcome Trust (grant 104924/14/Z/14), Deutsche Forschungsgemeinschaft (Research unit 1905 “Structure and function of the peroxisomal translocon”), and Oxford internal funds (EPA Cephalosporin Fund and John Fell Fund). Johnson Mak is recipient of funding from Australian National Health and Medical Research Council project grants App543107 and App1121697, and is a sub-awardee of National Institute of Health NIH RO1 GM064347. CF, JC, CE and DM designed the study. DM, CF, JC, PM and NY performed the experiments. JM provided the recombinant myr(-)Gag protein. CF developed the confinement index analysis. CF, JC and CE analysed the STED-FCS data. JC, CF, DM and CE wrote the manuscript. All authors contributed indiscussing the data, experiments and the manuscript.