Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.


Plasma membrane isolation from HIV-1 infected and uninfected MT-4 cells. For comprehen-
sive lipidome analysis, PMs were isolated from uninfected and infected MT-4 cells. Infected MT-4 cultures were >75% infected, as assessed by HIV-1 CA (capsid protein) expression ( Supplementary Fig. S1A). Since we were specifically interested in phosphoinositides, which are highly dynamic and sensitive to physical membrane alterations 37 , we selected a rapid method for PM isolation that does not actively alter the cell surface rather than applying the previously used blebbing or silica bead-based PM isolation methods 11,12 . PMs were separated from other cellular constituents by mechanical disruption of cells followed by a combination of differential and density centrifugations. Equal amounts of the different fractions obtained relative to input material were subjected to Western Blot analysis and the loss of organelle marker proteins within the different fractions was evaluated (Fig. 1). To assess the purity of the PM fraction, we determined enrichment of PM and removal of non-PM membranes by probing input and PM fractions with antibodies against marker proteins indicative of the presence of PM (sodium potassium ATPase (Na + /K + ATPase), TfR), endoplasmic reticulum (ER; calnexin), Golgi (GM130) and mitochondria (p30). Na + /K + ATPase and Tfr were clearly enriched in the PM fraction when compared to organelle membrane fractions, while the abundance of marker proteins for Golgi, ER and mitochondria was decreased in the PM fraction. To quantify these differences, we measured band intensities of the Western Blot shown in Fig. 1 and calculated the ratio between one of the PM markers (Na + /K + ATPase or Tfr) and each individual organelle marker (ER/Golgi/mitochondria) in the input and PM fractions. The two different PM markers were enriched 1.8-fold (+/− 0.02) compared to an ER marker in the PM fraction, while Golgi markers were decreased fourfold (+/− 0.03) and mitochondria markers were decreased 14-fold (+/− 0.12) relative to PM markers in PM isolations compared to input. This analysis indicated strong PM enrichment, while residual ERand a lower amount of Golgi membranes appear to be retained in the isolated PM fraction. ER contamination of PM isolations may cause some underestimation of PM-specific lipids in the PM, including e.g. phosphoinositides. Importantly, this method allowed isolation of PM with sufficient purity in a comparatively short period of time (the complete procedure takes less than 2 hours) at 4 °C.

Virus preparation and lipid extraction.
HIV-1 particles were purified from the medium of infected MT-4 cells by velocity gradient centrifugation on Optiprep gradients. This protocol yielded largely vesicle-free virus preparations 38 . Silver-stained gels of purified particles produced from MT-4 cells showed the Gag-derived CA and MA proteins as major HIV-1 particle constituents and confirmed the purity of the preparations and complete maturation ( Supplementary Fig. S1B). Virus stocks were further quantitatively analyzed for HIV-1 CA and genomic RNA content ( Supplementary Fig. S1C), and infectivity was titrated on susceptible target cells. These results allowed calculating the viral particle concentration in the respective samples and provided the basis for the subsequent estimation of viral lipid species on a per particle basis.
Standard lipid extraction according to Bligh and Dyer was applied as described 39 to recover all lipid species (except phosphoinositides) directly from whole cells, isolated PMs and virus preparations.
For phosphoinositide analyses, samples were TCA precipitated and then sequentially subjected to single-phase neutral and acidic extractions as shown in the schematic in Supplementary Fig. S2A. Internal standards were spiked into both extracts. Subsequently, both extracts were processed by two-phase acidic extraction ( Supplementary Fig. S2A). Lipids from neutral and acidic extracts were separately collected, derivatized, measured, and normalized based on the internal standards (standard curves for phosphoinositide quantitation are shown in Supplementary Fig. S2B-D). Finally, the normalized values from both extracts were summed up to yield the total level of the respective lipid in the original sample ( Supplementary Fig. S2E). This multistep procedure was necessary for quantitative recovery of the different phosphoinositides: while 70% of PI was observed in the neutral extract, PIPs were mainly present in the acidic extract and PIP 3 was exclusively recovered from the acidic extract ( Supplementary Fig. S2E), as had been observed for radioactively labeled phosphoinositides 40 .
Purified PM and virus samples contain little biological material compared to whole cell samples, which decreases the precipitation efficiency by TCA, and also the reproducibility of the two-step lipid extraction procedure due to lack of a visible pellet prior to neutral extraction. Therefore, we tested bovine serum albumin (BSA) and poly-D-lysine (PL) for their suitability to be used as a carrier during TCA precipitation of the purified PM and virus samples. For this purpose, we analyzed the recovery of PIP and PIP 2 in neutral and acidic extracts in the presence of BSA or PL by thin layer chromatography (TLC). PIP or PIP 2 standards were transferred into test tubes and dried, PL or BSA was added and the samples were recovered by TCA precipitation. Pellets were subjected to neutral and acidic extraction and extracts were run on TLC and stained with iodine vapors. In the presence of BSA, PIP and PIP 2 were lost from acidic extracts, since BSA remained soluble in the single-phase neutral extraction and precipitated at the interphase during two-phase extraction, leading to retention of PIP and PIP 2 in the neutral extract that contains the bulk of membrane lipids ( Supplementary Fig. S2F, BSA-samples). However, PL did not dissolve in the neutral extraction solvent and therefore turned out to be a suitable carrier with which we observed good recovery of PIP and PIP 2 in acidic and neutral extracts ( Supplementary Fig. S2F, PL-samples and S2G). 3% of each fraction (or 15% in lanes marked with "x5") were loaded. The membrane was probed with antibodies against marker proteins for PM (Na + /K + -ATPase and transferrin receptor (Tfr)), Golgi apparatus (GM130), ER (Calnexin) and mitochondria (p30) in three consecutive rounds (up to two antibodies, from different species, per round). For improved clarity and conciseness, cropped areas of the blot are shown. All cropped regions originate from the same blot.
www.nature.com/scientificreports www.nature.com/scientificreports/ Phosphoinositides purified by lipid extraction without further modification have low ionization efficiency due to their highly acidic nature, and thus yield large amounts of ambiguous ions during electrospray ionization. To improve their ionization efficiency, we employed a derivatization technique using TMS-diazomethane to methylate phosphate groups of phosphoinositides as previously described 36,41 . This neutralizes the phosphoinositides and therefore improves their ionization. The derivatized phosphoinositides are also more stable and volatile than their free acid forms 36 . This procedure enabled us to resolve the neutralized phosphoinositides based on their fatty acid chain composition during liquid chromatography.
Accordingly, isolated PMs and virus preparations were TCA-precipitated in the presence of PL for phosphoinositide analysis and subsequently subjected to two-step neutral and acidic lipid extraction, followed by permethylation. Lipid extracts from purified HIV-1, from isolated PM fractions from uninfected or HIV-1 infected MT-4 cells or from complete membrane fractions of uninfected or HIV-1 infected MT-4 cells were subjected to nano-electrospray ionization or liquid chromatography/electrospray ionization tandem mass spectrometry. Samples were analyzed for the different cellular lipid classes and lipid species. This led to quantitative determination of a total of 478 individual lipids, for the first time including the molecular species distributions of all three phosphoinositide classes.

Lipid composition of purified virus and producer cell membranes.
Comparison of the lipid composition and species distribution between whole cell extracts from uninfected and infected MT-4 cells revealed no major differences (see Supplementary Table 1 and Supplementary Fig. S3), consistent with previous reports suggesting that HIV-1 infection does not alter the overall lipid profile of target cells.
In general, we also did not observe significant differences when comparing the lipid composition of PMs from uninfected and HIV-1 infected MT-4 cells. The only exceptions were PIP 3 and triacylglycerol (TAG) levels, which were increased about 2-fold in the PMs of infected cells ( Fig. 2A, right panel and 2D). Since TAG is not a membrane lipid it can be assumed that TAG amounts were caused by contamination with non-bilayer lipids. Figure 2A shows that the phospholipid composition of HIV-1 membranes differs significantly from the host cell PM, as reported in previous studies 5,12 . PC, PE and PI levels were decreased in viral particles compared to host cell PM; the most abundant membrane phospholipid PC was reduced by half from ca. 20 mol% to ca. 10 mol% and PI levels were reduced even further from 5.2 mol% in the PM to 1.0 mol% in the viral membrane. In addition, the minor phospholipids lyso-PC (LPC) and phosphatidic acid (PA) were also reduced in the viral compared to the host cell PM; these two lipids had not been quantified in previous studies of HIV-1 membranes. www.nature.com/scientificreports www.nature.com/scientificreports/ PS was significantly enriched in HIV-1 when compared to the host cell PM as previously reported 5,12 . All phosphoinositides were detected in significantly higher amounts in viral particles when compared to the host cell PM ( Fig. 2A, middle and right panel, further discussed below). Also, sphingolipids differed between viral particles and PM isolations (Fig. 2B). While SM was highly enriched in HIV-1 particles, as reported 5,10-12 , ceramide (Cer) was strongly decreased. Dihexosylceramide (Hex2Cer) showed a slight decrease in HIV-1 particles. Consistent with HIV-1 budding from nanodomains enriched in sphingolipids and cholesterol, we found cholesterol to be clearly enriched in HIV-1 membranes when compared to PMs of infected and non-infected cells (Fig. 2C), as reported before 5,11,12 .
To determine the molecular composition of the HIV lipid envelope, we normalized the lipid molecule numbers to the number of viral particles present in the extract, which was calculated from the copy number of genomic viral RNA ( Supplementary Fig. S1C). This calculation yielded estimates for the total lipid content and for the number of molecules of different lipid classes in the membrane of an average HIV-1 particle. We calculated 430,000 lipid molecules per average virion based on four independent virus preparations. A graphical representation of the lipid composition of HIV-1 particles and numbers of respective lipid molecules per average HIV-1 particle for the various lipid classes are shown in Fig. 3B and Supplementary Table 2. We estimated approx. 2,000 PIP molecules, 7,800 PIP 2 molecules and 20 PIP 3 molecules in the lipid envelope per average HIV-1 particle. All three phosphoinositide classes were enriched when compared to the PM of HIV-1 infected MT-4 cells. Even though the absolute number of PIP 3 molecules per virion is low, PIP 3 is highly enriched in the HIV-1 lipid envelope as compared to the donor PM ( Fig. 2A).
We then determined the molecular species distribution of all phospholipids analyzed. Generally, acyl chain distributions were very similar in lipids from the viral membrane and from the producer cell PM ( Supplementary  Figs. S4-9). An exception were the molecular species of PC, lysoPC (LPC) and PI, as described before 12 . PC, LPC and PI were among the most strongly reduced lipids in the viral membrane (Fig. 3A). Compared with MT-4-derived PMs, we additionally observed a clear shift towards fully saturated acyl and ether PC species at the expense of mono-und poly-unsaturated PC species (Fig. 4A,B).
This shift was most pronounced for the shorter 30:0, 32:0 and 34:0 PC species. LPC also showed a strong enrichment of fully saturated species with short acyl chains at the expense of mono-and polyunsaturated LPC species (Fig. 4C).
For PI, we observed a clear shift towards short-chain PI species at the expense of longer ones and a reduction of polyunsaturated PI species with an increase of saturated and mono-unsaturated PI species ( Supplementary  Fig. S4A). In addition to what was observed before, we detected a shift towards shorter and less unsaturated species of acyl and ether/odd-chain fatty acyl-containing PG species in viral particles when compared to PMs of uninfected or infected cells ( Supplementary Fig. S4C The molecular species distribution of phosphoinositides was also largely similar in HIV-1 membranes and PMs of producer cells (Fig. 5 Table 1). The most abundant molecular species of PIP, PIP 2 and PIP 3 were the polyunsaturated 38:4 species and the monounsaturated 36:1 species (Fig. 5), showing that the metabolism of phosphoinositides is highly interconnected. Longer acyl chain species such as 36:1 were enriched in PIP 2 and PIP 3 species in the HIV-1 membrane compared to PM at the expense of shorter acyl chain species. Taking into account the elevation of PIP 2 levels in released virions, the viral membrane contains 4.2 times more PIP 2 36:1 than PM from infected cells (0.44 mol% vs. 0.1 mol%).

Discussion
Here, we present a comprehensive, quantitative lipidome analysis of HIV-1 in comparison with isolated host cell PMs from HIV-1 infected and uninfected cells. We detected and quantified 25 different lipid classes, covering the vast majority of the membrane lipid spectrum, including in total 478 molecular lipid species. Thus, this work significantly expands the coverage of lipid species described previously by us and others 5,10-12 and provides a quantitative analysis of all phosphoinositides, including PIP 3 , a minor phosphoinositide, which was not quantified in HIV-1 or PMs before. Based on this analysis, we present approximate numbers for lipid classes including phosphoinositides per average HIV-1 particle.
Our comprehensive analysis of the phosphoinositide content of plasma membranes isolated from HIV-1 infected cells and of HIV-1 particles derived thereof revealed a strong enrichment of PIP 2 and PIP 3 in viral membranes. The enrichment of PIP 2 is in line with previously published data 11 and the specific role of this lipid during HIV-1 assembly. As PI(4,5)P 2 is the most abundant phosphoinositide in mammalian cells and comprises >99% of the PIP 2 pool in mammalian plasma membranes 37,42 , PI(4,5)P 2 is most likely the dominating isomer enriched in the viral particle as well. PI(4,5)P 2 has been shown to be essential for PM targeting of HIV-1 Gag and thus for virus assembly and release 24,26,31 . Replenishing PI(4,5)P 2 in PI(4,5)P 2 depleted PM led to rapid induction of Gag assembly at the PM and production of infectious virus 31 . Taken together with structural analyses revealing a PI(4,5)P 2 binding site in the MA domain of HIV-1 Gag, one might therefore assume roughly stoichiometric amounts of Gag polyproteins and PI(4,5)P 2 molecules in HIV-1 particles. This is clearly not the case, however. Using quantitative lipid mass spectrometry combined with estimation of HIV-1 particle number in analytes, we determined the number of PIP 2 molecules per average HIV-1 particle to be 7,834 ±2885. This translates into a more than threefold molar excess of PI(4,5)P 2 over the ca. 2,500 Gag molecules per average HIV-1 particle. As the immature Gag layer does not completely cover the inner surface of the viral membrane 43 , Gag recruitment of PI(4,5)P 2 may be even stronger. Alternatively, Gag-free zones could also accommodate PI(4,5)P 2 clusters, but this could not explain the strong molar excess of PI(4,5)P 2 over Gag in the viral membrane.
A strong enrichment of PIP 2 in viral membranes is in line with our previous observation that PI(4,5)P 2 is needed to maintain the nascent Gag lattice at the PM during later stages of assembly, since partially assembled Gag structures were lost from the PM upon PI(4,5)P 2 depletion 31 . This result suggested a dynamic equilibrium between membrane-inserted and MA-buried myristate moieties with concomitant transient interactions of Gag with PI(4,5)P 2 molecules at the assembly site since stable insertion of a large number of Gag-linked myristates into the membrane bilayer would be expected to retain the assembling Gag lattice at the membrane. Accordingly, several recent reports indicated that multiple surfaces on MA interact transiently and dynamically with one or more PI(4,5)P 2 head groups 30,44 . Retaining PI(4,5)P 2 in the viral assembly domain by these recurrent, transient Gag interactions would explain the observed high PI(4,5)P 2 concentration in the virion. Given that PI(4,5)P 2 is by far the most prominent phosphoinositide in the viral membrane, there appears to be no further metabolic conversion once PI(4,5)P 2 is confined in the budding virion.
HIV-1 membranes were enriched in cholesterol, PS and SM at the expense of PC, PE and PI. These results are in good agreement with previously published HIV-1 lipidomics data [10][11][12] . Contrary to our previous studies 10,12 , pl-PE was not enriched in viral particles, and the observed slight decrease in pl-PE (0.9-fold when compared to PM from infected cells) was similar to the report by Chan and colleagues 11 . These authors also reported an increase in cholesterol in the HIV-1 membrane in comparison to PM from infected cells 11 , which was confirmed here. The observed minor differences between the different lipidomics studies may have been caused by differences in cell types and membrane isolation methods. While Lorizate et al. and Chan et al. 11,12 used cationic colloidal silica beads to extract PMs, we used density gradient centrifugation. This method should not interfere with host cell metabolism, as we do not apply any chemical stimulation or alteration of the cell surface.
All studies of HIV-1 lipidomes reported a major increase of the outer leaflet lipid SM in the viral membrane [10][11][12] . In the current study, SM was enriched almost twofold in viral particles compared to PM from virus-producing cells. Since SM is restricted to the outer leaflet and Gag is a peripheral membrane-binding protein associating with the inner PM leaflet, Gag cannot directly recruit outer leaflet lipids into the HIV-1 assembly www.nature.com/scientificreports www.nature.com/scientificreports/ site. Obvious candidates for mediating Gag-dependent recruitment of SM in the outer leaflet would be inner leaflet lipids directly interacting with Gag or recruited by Gag into the assembly domain. Trans-bilayer coupling of inner and outer leaflet lipids has been reported to mediate actin dependent clustering of outer leaflet lipids and was shown to depend on acyl chain length of the respective lipids 45 . In this case, trans-bilayer coupling depended on PS 18:1/18:0 (i.e. 36:1), while molecular species of PS with shorter acyl chains could not mediate this effect 45 . Obvious candidates for inner leaflet lipids to couple outer leaflet lipids with HIV-1 Gag assembly would be PI(4,5) P 2 and PS, both of which were found to be enriched in viral membranes and to be recruited by Gag. HIV-1 particles showed fourfold enrichment in PIP 2 36:1 (0.44 mol% in particles versus 0.10% in infected PMs). In addition, a slight but significant 1.3-fold increase of PS 36:1 was observed in virions when compared to PMs of infected cells. These results are consistent with the reported involvement of PS 36:1 in coupling outer leaflet lipid clustering to cytoplasmic signaling 46 , and would suggest a similar mechanism of trans-bilayer coupling for Gag-induced PS 36:1 and possibly PIP 2 36:1 clustering. Even though we detected a 16-fold increase of PIP 3 36:1 in virions compared to producer cell PM, the levels of 36:1 PIP 3 in HIV-1 particles (0.0013 mol%) remain very low and a significant contribution to trans-bilayer coupling therefore seems less likely. Conceivably, the Gag-linked myristate group could also contribute to trans-bilayer coupling as has been observed for dimyristoylphosphatidylcholine in membrane bilayers 47 . However, in the case of HIV-1 Gag the relatively short myristate acyl chain (C14) is stably anchored on the Gag protein and is thus unlikely to reach sufficiently deep into the bilayer.
In summary, our results are consistent with HIV-1 budding from sphingolipid and cholesterol enriched nanodomains. The observed strong enrichment of PIP 2 in the viral membrane supports our previously reported www.nature.com/scientificreports www.nature.com/scientificreports/ hypothesis of transient Gag interactions with PM PI(4,5)P 2 , and concomitant flipping of myristate between the inner leaflet of the PM and a lipid-binding pocket in the MA domain of Gag 31 . PIP 2 and possibly also PS recruitment may then mediate accumulation of outer leaflet SM, thus rigidifying the viral membrane. Budding of HIV-1 from domains rich in sphingolipids and cholesterol might facilitate the fission step that releases viral particles from the plasma membrane by a mechanism that involves a tension gradient between more ordered domains in the viral bud and less ordered domains in the neck area 48 . This model would suggest Gag to be the master-regulator of the assembly membrane, directly or indirectly recruiting specific lipid species. While the alternative model of Gag targeting to pre-existing lipid microdomains of the observed composition and arranged by a different membrane regulator appears less likely, it is not excluded by the current results. Advances in multi-color super-resolution microscopy may in the future allow directly visualizing lipids and proteins at HIV-1 assembly sites, thus shedding light on this important issue.

Materials and Methods
Reagents. All chemicals and reagents were purchased from commercial sources unless otherwise noted. Plasmid pNL4-3 was described previously 49 .
For virus production, MT-4 cells 50 were infected with HIV-1 strain NL4-3 49 , and the virus was harvested from co-cultures of infected and uninfected cells before cytopathic effects were observed as described in 38 . HIV-1 purification was performed as described 38 . Briefly, particles were concentrated from cleared media by centrifugation through a cushion of 20% (w/v) sucrose in PBS. Concentrated HIV-1 was further purified by velocity gradient centrifugation on an Optiprep gradient (Axis-Shield, Oslo, Norway). The visible virus band was collected and pelleted yielding an 1,800-fold concentration compared with the initial volume. Purity was assessed by separation of particles by SDS-PAGE (12.5% acrylamide) and subsequent silver-staining. Virus production was quantitated by an in-house enzyme-linked immunosorbent assay (ELISA) detecting the HIV-1 CA protein p24 51 . Virus titers were determined by evaluation of RNA copy number with PCR, as follows.

Analysis of HIV-1 RNA copy number. Number of viruses in concentrated virus stocks was estimated
by analysis of RNA copy number. For this, virus stocks were diluted 1:10 with PBS and lysed with 2x lysis buffer (50 mM KCl, 100 mM Tris-HCl, 40% Glycerol, 0.25% Triton X-100; pH 7.4). Subsequently, lysed samples were stepwise diluted further with PBS to a final dilution of 1:10,000,000-1:40,000,000. 600 µl of the dilutions were subjected to quantitative PCR using the Abbott RealTime HIV-1 assay REF 6L18 (Abbott Molecular Inc., Des Plaines, IL, USA) and the assay was performed according to the manufacturer's instructions. The Abbott RealTime HIV-1 assay is able to quantitate HIV-1 over the range of 40 to 10,000,000 copies/ml and diluted samples measured here were in the range of approximately 60,000 to 200,000 copies/ml. Half of the resulting material was further used for general lipidomics and the other half for phosphoinositide lipidomic analysis. For this, samples were resuspended in PBS (general lipidomics) or water (phosphoinositide lipidomics). Samples used for phosphoinositide analysis were gently mixed with delipidated poly-D-lysine at a final concentration of 1 mg/ml. Afterwards, TCA was added to a final concentration of 10% (w/v), samples were vortexed for 30 s and incubated on ice for 15 min. Finally, samples were centrifuged at 20.000 g for 3 min at 4 °C, supernatant was discarded, and samples were frozen at − 80 °C Whole cells were harvested at the same time, washed with PBS and directly frozen at -80 °C as cell pellets (general lipidomics) or TCA precipitated (phosphoinositide lipidomics) with 10% ice-cold TCA in deionized water (as above but in the absence of poly-D-lysine), pelleted and frozen at -80 °C. www.nature.com/scientificreports www.nature.com/scientificreports/ Biosciences, Franklin Lakes, NJ, USA), polyclonal anti-Calnexin (RRID:AB_11178981; Enzo Life Sciences GmbH, Lörrach, Germany) and polyclonal anti-p30 (kindly donated by W. Just, BZH, Heidelberg) antibodies, respectively, followed by secondary antibodies coupled to IRDye 800 or IRDye 680 (RRIDs:AB_621847 and AB_621845; LI-COR Biosciences, Lincoln, NE, USA). Fluorescent signals were detected using a LI-COR Odyssey CLx scanning system. To quantify band intensities, blots were analyzed using the Odyssey Image Studio v5.2 software (LI-COR Biosciences, Lincoln, NE, USA).
Sample preparation prior to lipid extraction. All generated samples were split for general lipidomic and phosphoinositide measurements.
For general lipidomic analyses, cell pellets, PM isolations in PBS and purified HIV-1 in PBS were added to an excess of MeOH and subsequently subjected to lipid extraction.
For phosphoinositide lipidomic analyses, purified HIV-1 in PBS was mixed with 1 mg/ml delipidated poly-D-lysine and subjected to ice-cold TCA (10% (w/v) final concentration). TCA-treated cell pellets, PM isolations and HIV-1 particles were washed 2 x with 5% TCA + 10 mM EDTA. Subsequently, all samples were subjected to lipid extraction.
Lipid extraction and analysis of general lipids. Cells, PM and viral particles were subjected to lipid extractions using an acidic Bligh & Dyer, except from plasmalogens, which were extracted under neutral conditions 52 . Lipid standards were added prior to extractions, using a master mix containing phosphatidylcholine nano-electrospray infusion and ionization via a Triversa Nanomate (Advion Biosciences, Ithaca, NY, USA) as previously described 39 . Resuspended lipid extracts were diluted 1:10 in 96-well plates (Eppendorf twin tec 96, colorless, Z651400-25A; Sigma-Aldrich, St. Louis, MO, USA) prior to measurement. Lipid classes were analyzed in positive ion mode applying either specific precursor ion (PC, lyso-PC, SM, cholesterol, Cer, HexCer, Hex2Cer, and PE-P) or neutral loss (PE, PS, PI, PG, and PA) scanning as described in 39 .
Lipid extraction, derivatization and analysis of phosphoinositides. 720 µl of CHCl 3 :MeOH 1:2 were added to all TCA pellets and vortexed for 10 min at RT. Samples were centrifuged at max. speed for 5 min at RT. The supernatants were transferred into fresh tubes on ice, and regarded as neutral extracts, to which the ISD mix was spiked only after they were transferred into fresh tubes. Remaining pellets were stored under argon at -80 °C for acidic extraction.
For neutral extraction, 10 µl phosphatidylinositol lipid analytical internal standard (ISD) mix (see below for composition) was added to the supernatants, samples were vortexed and spun down briefly. 6 µl of ice cold 12.1 M HCl were added and samples were vortexed immediately within one second of addition for 10 min at 4 °C. 720 µl CHCl 3 were added to the samples and tubes were vortexed for 5 min at 4 °C, followed by addition of 354 µl 1 M HCl and subsequent vortexing for 2 min at 4 °C. Samples were centrifuged at 1000 g for 5 min. The lower phase was transferred into fresh tubes and dried in a refrigerated Centrivap (Labconco, Kansas City, MO, USA), while preventing air condensation. Dried tubes were flushed with argon and stored at -80 °C.
For acidic extraction, tubes containing the remaining pellets were placed on ice and 10 µl of the ISD mix (see below for composition) were added. 726 µl of CHCl 3 :MeOH:12.1 M HCl 40:80:1 were added and tubes were vortexed for 15 min at 4 °C. 720 µl CHCl 3 were added and tubes were vortexed for 5 min at 4 °C, followed by addition of 354 µl 1 M HCl and vortexing for 2 min at 4 °C. Samples were centrifuged at 1000 g for 5 min and the lower phase was transferred into fresh tubes. 702 µl of CHCl 3 :MeOH:1.185 M HCl 3:48:47 (theoretical upper phase) were added to the samples and samples were vortexed for 10 s, followed by centrifugation at 1000 g for 3 min at www.nature.com/scientificreports www.nature.com/scientificreports/ to each hydroxyl on the phosphate groups of phosphoinositides. The reaction takes place in the presence of methanol, which acts as proton donor and removes TMS as a methyl ether, after which a reactive intermediate is formed that methylates one hydroxyl on a phosphate group while releasing N 2(g) as by-product 54 . Briefly, the dried lipids were redissolved in 100 uL of methanol:dichloromethane:TMS-diazomethane (2.0 M solution in Et 2 O) (4:5:1) by vortexing. This resulted in a final TMS-diazomethane concentration of 0.2 M. The permethylation reaction started as soon as the solution was added, and the reaction was continued for 40 min at RT. Samples were dried in a CentriVap (Labconco) with a carefully set vacuum control. The dried samples were flushed with argon and stored at − 80 °C prior to mass spectrometric analysis.
The dried, derivatized samples were redissolved in methanol, and quantified by targeted analysis as described previously 41 . Briefly, dried samples were initially re-suspended in 20-100 µl 100% methanol (LC-MS Optima grade, Fisher) prior to chromatographic separation at ambient temperature using a C4 column (Waters Acquity UPLC Protein BEH C4, 1.7 µm 1.1 × 100; 300 A). A Waters Acquity FTN autosampler set at 4 °C injected 1-3 µl of sample via Waters Acquity UPLC. For chromatography of phosphoinositides the mobile phase was delivered over an 18.5 min runtime at a flow rate of 0.1 ml/min by a Waters Acquity UPLC. The gradient was initiated with 10 mM formic acid in water/10 mM formic acid in acetonitrile (37:63 v/v), held for 2 min, then increased to 15:85, v/v in 10 min, then increased to 100% B and held at 100% for 2.8 min prior to 3 min re-equilibration to starting conditions. The gradient was initiated with 10 mM formic acid in water/10 mM formic acid in acetonitrile (45:55 v/v), held for 2 min, then increased to 15:85, v/v in 10 min, then increased to 100% B and held at 100% for 1.8 min and then re-equilibrated to starting conditions for 3 min.

Statistical analysis. Statistical analysis was performed with supervision of the Heidelberg Hospital Medical
Biometry and Informatics (IMBI) Department. Statistics are given as mean ± SD. Student's t-test was used to test for statistical significance. P-values are given in figure legends.