Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions

Abstract

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.

Fig. 1.

Nef increases the synthesis of cholesterol in cells. (a) Nef does not affect the uptake of exogenous cholesterol. Three CHO cell lines, K1, MET18B-2, and 215 cells, were transfected with pcDNA (white bars) or pcDNA-Nef (black bars). Total cellular cholesterol was quantified by an enzyme-linked assay. Levels of cholesterol were normalized to amounts of total cellular protein. (b) Detection of newly synthesized cholesterol. CHO cells were labeled with radioactive cholesterol precursor, [³H]mevalonic acid. Newly synthesized labeled cholesterol was visualized by autoradiography following TLC. The top arrow denotes cholesterol and the lower arrow denotes 4-carboxysterol. (c) Nef increases the synthesis of cholesterol in CHO cells. CHO K1 cells, which expressed the empty plasmid vector (0, white bar), wild-type (black bar), or mutant myristoylation-negative NefG2A (striped bar) proteins were labeled with [³H]mevalonic acid. Newly synthesized labeled cholesterol was quantified using a scintillation counter and normalized to levels of total cellular protein. (d) Nef increases the synthesis of cholesterol in Jurkat cells. Newly synthesized cholesterol from Nef-inducible Jurkat cells, which were labeled with [³H]mevalonic acid, was quantified as above. In all experiments, the expression of Nef was confirmed by Western blotting, which is denoted by arrows in a, c, and d, Lower. Standard deviations from the mean reflect three independent experiments.

Fig. 2.

Nef increases the expression of the CYP51 gene in Jurkat cells. (Top) The CYP51 promoter directed the expression of the luciferase reporter gene and is presented schematically. (Middle) Luciferase activity was determined in cell lysates of Jurkat cells. (Bottom) The expression of Nef proteins, denoted by an arrow, was confirmed by Western blotting. Standard deviations from the mean are representative of three independent experiments.

Fig. 3.

Nef increases levels of newly synthesized cholesterol in lipid rafts and HIV particles. (a) Isolation of DRMs from cells. 293T cells were transfected with a CD45-expression plasmid, and three fractions were isolated: DRM, cytosol, and plasma membrane (PM). They were analyzed with the cholera toxin B, which detects GM1, or the anti-CD45 monoclonal antibody by Western blotting. (b) Distributions of the wild-type Nef and mutant NefG2A protein in different fractions. 293T cells were transfected with three HIV proviruses, the wild-type (HIV WT), and those lacking Nef (HIV ΔNef) or expressing Nef without the myristoylation site (HIV NefG2A), respectively. Cells were then fractionated, and different fractions were analyzed with the cholera toxin B or the anti-Nef monoclonal antibody by Western blotting. (c) Nef increases levels of newly synthesized cholesterol in lipid rafts. 293T cells were transfected with three proviruses as in b and labeled with [³H]mevalonic acid. DRMs were isolated and quantified by scintillation counting. The radioactivity was normalized to levels of total cellular protein. Bars represent the following: white, HIV ΔNef; striped, HIV NefG2A; black, HIV Nef. (d) Nef increases levels of newly synthesized cholesterol in HIV particles from 293T cells. Viruses, collected from the 293T cells and labeled with [³H]mevalonic acid, were purified, and labeled cholesterol isolated from these particles was quantified by scintillation counting. Radioactivity was normalized to levels of p24^Gag (Top). The expression of Nef and Gag proteins from these viruses was confirmed by Western blotting (Middle and Bottom). NefΔN was cut by the viral protease and lacks the N terminus of Nef. (e) Nef increases levels of newly synthesized cholesterol in HIV particles from Jurkat cells. As in d, levels of newly synthesized cholesterol, which were normalized to amounts of p24^Gag, are presented (Top). The expression of Nef and Gag in these viruses, confirmed by Western blotting, is presented (Middle and Bottom) and denoted by arrows. Standard deviations from the mean are from three independent experiments (c–e).

Fig. 4.

Nef binds cholesterol in vitro and in vivo.(a) A putative CRM (37) in Nef. The upper sequence represents the C terminus of wild-type Nef from HIV-1_SF2. The lower sequence contains the mutations introduced to disrupt this motif. (b) Expression of recombinant Nef proteins from E. coli. Wild-type Nef and mutant NefCRM proteins were expressed in the pET system and purified by His Bind Resins. The purity of these proteins is demonstrated by SDS/PAGE followed by Coomassie blue staining (Left). Western blotting (WB), using polyclonal (pAb) and monoclonal (mAb) antibodies against Nef, is presented (Center and Right, respectively). Arrows denote different Nef proteins. (c) Nef binds cholesterol in vitro. Binding of cholesterol to purified Nef proteins was determined after incubating them with photoactivatable [17α-methyl-³H]promegestone in the absence and presence of increased levels of cold cholesterol. Labeled bands were visualized by autoradiography after SDS/PAGE. Arrow denotes Nef with bound [³H]promegestone (Bd, beads alone). (d) Nef binds cholesterol in vivo. 293T cells expressed truncated CD8T, which contains the extracellular and transmembrane domains of CD8 (white bar), wild-type CD8.Nef (CD8T fused to Nef, black bar), and mutant CD8.NefCRM (CD8T fused to NefCRM, cross-hatched bar) chimeras. Cells were then labeled with the photoactivatable [17α-methyl-³H]promegestone and immunoprecipitated with the anti-CD8 antibody. The expression of these proteins was confirmed with anti-Nef and CD8 antibodies (Lower). Levels of [17α-methyl-³H]promegestone on immunoprecipitated proteins were quantified with scintillation counting (Upper). Standard deviations from the mean are from three independent experiments (d).

Fig. 5.

CRM is required for Nef to increase HIV infectivity. (a) Increased levels of newly synthesized cholesterol in lipid rafts and viral particles by Nef depend on the CRM. 293T cells were transfected with four different proviruses expressing the wild-type Nef (HIV WT, black bar), mutant NefG2A protein (HIV NefG2A, striped bar), no Nef (HIVΔNef, white bar), or mutant NefCRM protein (HIV NefCRM, cross-hatched bar). Cells were labeled with [³H]mevalonic acid. Labeled cholesterol from total cell lysates, DRM, and viral particles was extracted, quantified by scintillation counting, and normalized to levels of cellular protein or p24^Gag. Results are representative of three independent experiments. (b) Cholesterol binding to Nef increases HIV infectivity. GHOST indicator cells were infected with equal amounts of the same viruses as in a. Two days later, the expression of GFP in infected cells was quantified by flow cytometry. Results presented are representative of three independent experiments. (c–e) Expression levels of Gag and Nef in transfected cells (c) and virions (d) and of Nef in DRM were determined by Western blotting.

Fig. 6.

A model for the effects of Nef on viral infectivity. Nef is expressed as an oligomer abundantly before viral structural proteins. It activates cellular signaling cascades and causes cytoskeletal rearrangements (step 1). These lead to increased transcription of at least one cholesterogenic enzyme, CYP51 (step 2). Increased CYP51 activity (blue circles) increases synthesis of cholesterol (red circles) in the endoplasmic reticulum (step 3). The model shows the transport of Nef in viral assembly intermediates (step 4) to lipid rafts (step 5). Nef and newly synthesized cholesterol are incorporated into DRM and virions (step 6). More infectious viral particles are released into extracellular space (step 7). Nef is also cleaved by the viral protease in virions (step 7).