Retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides

Abstract

Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P(2) was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P(2) to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P(2) interactions for particle release from cells.

FIG. 1.

Lipid composition of retroviruses in comparison to the lipid composition of total membranes of producer cells. (A) Lipid composition of different retrovirus envelopes and microvesicles (MV) produced from various cell types as described in the text. Values are expressed as molar percentages of a given lipid to the total lipid measured (except cholesterol and GM3, which were normalized to the total PC and SM signal intensities, respectively). PA, glycerophosphates; n/a, not available. (B) Ratio of retroviral lipids to total membrane lipids of host cells. Lipids that are significantly enriched (>1.5-fold) or reduced (<1.5-fold) in viral envelopes are highlighted in red and green, respectively. Each experiment was performed at least three times (n ≥ 3). Values that were significantly different (P < 0.05) from total membranes of producer cells are indicated by an asterisk.

FIG. 2.

The lipid composition of retroviruses resembles that of plasma membranes. (A) Plasma membrane (PM) fractions were isolated using cationic beads. Western blots with sera against raft (flotillin and caveolin) and nonraft markers (transferrin receptor [TrR]) were used to assess the quality of the plasma membranes, while actin (cytoplasmic protein) and Rab5 (endosomal protein) served as indicators for the purity of the plasma membranes. Conditions equivalent to 1% bead solution were used for the determination of lipid contents. TM, total membranes; −, no beads. (B to G) Time of flight ESI-MS (negative mode) are shown for lipid mixtures from MLV (B), REF plasma membrane (D), and REF total membranes (TM) (F). The lipids extracted from CD45-depleted HIV (C), H9 plasma membrane (E), and H9 total membranes (G) were analyzed similarly. The representative spectra shown are normalized to the highest peak within the m/z range. Prominent ions which were characterized by tandem MS are labeled. (H) Enrichment of phosphoinositides in retroviral envelopes. Precursor ion scanning for m/z 241 (dehydrated inositol fragment) was used to detect PI and phosphoinositides in MLV and plasma membrane.

FIG. 3.

Quantification of individual species of glycerophospholipids and sphingolipids of HIV and H9 host cells. Abundance is represented as the molar percentages (y axis) of a given lipid (x axis) to total lipid measured, except for GM3, which was normalized to the total SM levels. Lipids were extracted from purified virus (black bars), total cell membrane, and plasma membrane fractions and quantified via MS using multiple reaction monitoring. The percentages were calculated with relevant internal standards. GM3 quantification is represented in relative levels due to the lack of suitable internal standards. Sphinogolipids are presented as sphinogoid base residue/fatty acyl residue.

FIG. 4.

Lipid composition of retroviruses in comparison to the lipid composition of the plasma membranes of producer cells. (A) Values are expressed as molar percentages (except for cholesterol and GM3). (B) Ratio of retroviral and microvesicle (MV) lipid composition to plasma membrane lipid composition. Lipids that are significantly enriched (>1.5-fold) or reduced (<1.5-fold) in viral envelopes are highlighted in red and green, respectively. Each experiment was performed at least three times (n ≥ 3). Values that were significantly different (P < 0.05) from plasma membrane fractions of producer cells are indicated by an asterisk.

FIG. 5.

Effects of PI(4,5)P₂ depletion on HIV (A) and MLV (B) release from HEK293 cells. The effect of transient expression of 5PaseIV and 5PaseΔ1 was determined by normalizing virus infectivity released from samples transfected with empty vector and presented as inhibition of infectious virus released. The ratio data were obtained by normalizing the 5-phosphatase IV (5PaseIV) to 5PaseΔ1 values. The Western blots show the levels of HIV (A) and MLV (B) Gag expression in cells and released virus detected in culture supernatants.