Human immunodeficiency virus type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

Abstract

Background: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition.

Results: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells.

Conclusion: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

Figure 1

Nef boosts HIV-1 infectivity and replication without increasing microdomain association of Gag in producer cells. (A) Single round of replication analysis on TZM cells. TZM cells were infected with 0.5 ng CA of the indicated virus stocks. 36 hours post infection, the cells were fixed, stained for β-galactosidase activity and the number of blue cells was counted. Data represent average values from three independent experiments with triplicate measurements each with the indicated standard error of the mean. Depicted is the relative virion infectivity (number of blue cells per ng CA) with values for HIV-1_NL4-3 NefSF2 (wt) arbitrarily set to 100%. (B) HIV-1 replication in PBL. HIV replication was measured in 96 well plates on 1 × 10⁵ PBL per well and 1 ng CA virus input. Freshly isolated, non-activated cells were infected (day -6) for three days and subsequently activated by PHA/IL-2 for three days. Starting from day 0, cells were kept in the presence of IL-2 and cell culture supernatants were collected each day to monitor CA production. CA values represent the average from quadruplicate infections performed in parallel. (C-D) Lipid raft flotation analysis from infected MT-4 (C) or transfected Jurkat T lymphocytes (D). Cell lysates (1% Triton X-100) were separated by Optiprep gradient ultracentrifugation, and eight fractions were collected from the top (fraction 1) to the bottom (fraction 8) of the gradient. The detergent resistant membrane fraction (DRM, fraction 2) and the pooled nonraft (soluble) fractions (S, fractions 7 and 8) were analyzed together with the unfractionated cell lysate (L) by Western Blotting for the distribution of Gag (top), Nef (middle) and TfR (bottom).

Figure 2

Characterization of purified HIV-1 particles. HIV-1 virions were purified from cell culture supernatants (see Materials and Methods for details). (A), Western Blot and silver stain analysis of the indicated virion preparations for major viral particle constituents. (B) Single round of replication analysis on TZM cells with the particle preparations analyzed in A. The assay was performed analogous to that described in Fig. 1A. (C) Relative amounts of total cell culture supernatant p24 recovered after the optiprep procedure. Depicted are average p24 amounts recovered in the virion preparation procedure relative to the total input from four independent purifications with the indicated standard error of the mean.

Figure 3

Lipid analysis of virus particles. Quantitative lipid analysis was performed as described in Methods. Data are displayed as molar ratio of individual lipid classes to PC. Values present the average from at least three independent experiments with error bars indicating the standard deviation of the mean. (A) PC molecular species distribution given in % of total. (B) Number of species in % of total either containing none, one, or two or more than two double bonds in both fatty acids. (C) Analysis of all PC species. X:Y values on the x-axis denote the total number of C- atoms of both fatty acids (X) and the total number of double bonds (Y), respectively.

Figure 4

Nef does not affect the cholesterol content of HIV-1 particles. (A) Quantitative lipid analysis was performed as described in Methods. Data are displayed as molar ratio of cholesterol to PC. Error bars represent standard deviation of the mean. (B) Selected high-field region of the¹H spectra of 0.4 mM Nef-SF2 (Δ_1–43, C210A) in the absence (black) and presence of 0.4 mM water soluble cholesterol (red), in comparison with water soluble cholesterol dissolved in the same buffer as the protein (green).(C) ¹H,¹⁵N-TROSY-HSQC spectra of 0.4 mM Nef-SF2 (Δ_1–43, C210A) highlighting an overlay of the C-terminal residues including the putative cholesterol binding site in the absence (black) and presence of 0.4 mM water soluble cholesterol (red) or 0.4 mM methyl-β-cyclodextrine (green).

Figure 5

Elevation of SM/PC ratios is insufficient for Nef-mediated enhancement of virion infectivity. (A) Single round of infection analysis on TZM cells with cell culture supernatants from MT-4 T lymphocytes infected with the indicated viruses. The assay was performed analogous to that described in Fig. 1A. (B) Quantitative lipid analysis of virion SM/PC ratios. HIV particles purified from the cell culture supernatants analyzed in A were subjected to lipidome analysis. Depicted are the SM to PC ratios.

Figure 6

Nef enhances virion infectivity independently of whether HIV-1 entry occurs via membrane microdomains. Single round infection analysis on HeLa cells transiently expressing the indicated CD4 variants. Productively infected, p24CA positive cells were quantified 36 hours post infection by flow cytometry. Data represent average values from three independent experiments with the indicated standard error of the mean. Depicted is the relative virion infectivity with values for HIV-1_NL4-3 NefSF2 (wt) arbitrarily set to 100%.

Figure 7

Nef alters the lipid composition of DRMs in HIV-1 infected T lymphocytes. DRM flotation analysis was performed from MT4 cells infected with the indicated viruses as described for Fig. 1D and quantitative lipid analysis was performed from L, S and DRM fractions. Depicted are the SM to PC ratios.