The HIV lipidome: a raft with an unusual composition

Abstract

The lipids of enveloped viruses play critical roles in viral morphogenesis and infectivity. They are derived from the host membranes from which virus budding occurs, but the precise lipid composition has not been determined for any virus. Employing mass spectrometry, this study provides a quantitative analysis of the lipid constituents of HIV and a comprehensive comparison with its host membranes. Both a substantial enrichment of the unusual sphingolipid dihydrosphingomyelin and a loss of viral infectivity upon inhibition of sphingolipid biosynthesis in host cells are reported, establishing a critical role for this lipid class in the HIV replication cycle. Intriguingly, the overall lipid composition of native HIV membranes resembles detergent-resistant membrane microdomains and is strikingly different from that of host cell membranes. With this composition, the HIV lipidome provides strong evidence for the existence of lipid rafts in living cells.

Fig. 1.

Lipid composition of MT-4 cells and HIV-1. Quantitative lipid analysis was performed as described in Materials and Methods. Data are displayed as molar ratio of individual lipid classes to PC. Error bars represent standard deviation of the mean.

Fig. 2.

Enrichment of DHSM 16:0/PC in HIV-1. (a) Lipids of cells (Upper) and virus (Lower) were subjected to mass spectrometer analysis. Precursor ion scanning was performed by selecting for fragment ions of m/z 184 Da. Major peaks are labeled giving lipid class, number of total C atoms, and double bonds in acylated fatty acids. Spectra are normalized to the highest peak in the displayed mass range. (b) Quantitative analysis of SM and DHSM was performed as described in Materials and Methods. Data are displayed as molar ratios of the indicated lipid species to PC. Ratios determined for host cell membranes were set to 1. Error bars represent standard deviation of the mean.

Fig. 3.

FB1 treatment of virus-producing cells reduces HIV-1 infectivity. HIV-1 infected MT-4 cells were cultured in the absence or presence of 50 μM FB1. (a) Quantitation of lipids from purified virus was performed by nano-electrospray ionization tandem mass spectrometry. Values are expressed as molar ratios of individual lipid classes to PC. (b) Quantitation of HIV-1 release (measured by antigen ELISA; Left) and infectivity of cell-free virus (measured by infection of TZM cells; untreated samples were set to 100%; Right). Bars 1 and 2 correspond to two independent infections, each performed in triplicate. Error bars represent standard deviation of the mean.

Fig. 4.

DRM from infected cells and native HIV-1 membranes show similar enrichment of SM and DHSM. HIV-1 particles and DRM were isolated as described in Materials and Methods. The SM, DHSM, and PC content in virus membranes and DRM was determined by nano-electrospray ionization tandem mass spectrometry, whereas the distribution of selected proteins was determined by Western blot. (a) DRM obtained by cold extraction of HIV-1 infected MT-4 cells with Triton X-100 (TX100, Upper) or Brij 96 (Lower) were analyzed for raft (flotillin 1) and nonraft (transferrin receptor, TfR) marker distribution. (b) SMs to PC ratios in total cell membranes, HIV-1, and DRM obtained with TX100 or Brij 96. (c) SM (16:0) and DHSM (16:0) to PC ratios in HIV-1 and DRM. (d) Extracts from infected MT-4 cells (lane 1), DRM from infected cells (obtained by cold TX100 extraction; lanes 2–4), and purified HIV-1 (lanes 5–7) were subjected to Western blot analysis detecting flotillin or gp41. The amount of cell extracts loaded was normalized according to cell number (10⁵ cells in lane 1; DRM from 3 × 10⁵, 2 × 10⁵, and 10⁵ cells in lanes 2, 3 and 4, respectively), and the amount of virus loaded was normalized according to total PC and SM content of DRM and HIV-1 preparations with equal amounts of lipids loaded in lanes 4 and 6 (lane 5, 500% of lane 6; lane 7, 50% of lane 6). Error bars represent standard deviation of the mean.

Fig. 5.

The lipid composition of HIV particles. For details, see text.