Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV‐1 Env Clustering

Abstract HIV‐1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751–854 in the cytoplasmic tail (CT751–854). Super‐resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751–854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.


Lipid incorporation.
Lipid incorporation yield was studied in HEK 293T seeded in 35 mm dishes. 10 μCi/dish of [ 3 H]-photochol was added in 2 mL DMEM GlutaMAX TM High Glucose culture medium supplemented with 10% delipidated or lipidated FBS, and 100 U/ml Penicillin-Streptomycin, and incubated with the cells for different times. Cells were then scraped, pelleted, and resuspended in 100 μL of methanol and 10 μL of water, and vortexed vigorously. The samples were centrifuged at 14,500 rpm for 15 min to separate the aqueous and organic phases. The upper organic phase containing the extracted lipids was collected, and radioactivity of the sample (in disintegrations per minute or DPM) was determined by a Tri-Carb 2900TR (Perkin Elmer) scintilliation counter. The radioactive signal in μCi was obtained from the DPM data with the following formula: 1 µCi = 2.2 x 10 6 DPM To determine the lipid incorporation into the virus, viral particles were purified by Optiprep gradient and their lipids were extracted as explained above and the radioactivity of the sample was determined by a scintilliation counter. To calculate the number of [ 3 H]-photochol in each viral particle, the CA amount of the sample was quantified by an anti-CA Western blot, and assuming ~2500 molecules of CA [3,4] and ~80,000 molecules of chol [4,5] in each viral particle, the percentage of incorporated [ 3 H]-photochol compared to total chol was calculated.

Chessie-8 coupling to beads.
For gp41 immunoprecipitation the anti-gp41 chessie-8 antibody was covalently coupled to Protein G Sepharose 4 Fast Flow (GE Healthcare) beads, so that the heavy chain of that the antibody, with a similar size to gp41, does not interfere in the Western blot detection of the Detection of the proteins was carried out using the LI-COR Odyssey imaging system. The intensity signal of the protein band corresponding to gp41 was plotted against the loaded ng of CA, and a linear regression curve was obtained. The slopes of the linear regression curves of the variants (corresponding to the gp41/CA ratio) were compared to the wild-type particles to calculate the relative gp4 content of the viral particles.

Lipidomic analysis.
Lipids were extracted with a methanol-chloroform (1:2, vol/vol) solution containing internal standards (N-dodecanoylsphingosylphosphorylcholine, 16:0 D31_18:1 phosphocholine, 0.2 nmol each, and 2 nmol of stigmasterol from Avanti Polar Lipids). Extracts were evaporated and solved in MeOH before analysis. Lipids were analysed by liquid chromatography-high resolution mass spectrometry using an Acquity UHPLC BEH C8 column and an Acquity ultra high-performance liquid chromatography (UHPLC) system (Waters, USA) connected to a Time of Flight (LCT Premier XE) Detector. Positive identification of compounds was based 4 on the accurate mass measurement with an error B5 ppm and its LC retention time, compared with that of a standard (92%).

Proteomic analysis.
Proteins detected in the gp41 immunoprecipitation were subjected to proteomic analysis by mass spectrometry. gp41 was immunoprecipitated as before from purified viral particles and loaded into an SDS-PAGE. The gel was then stained with Coomassue blue, and the bands of an apparent ~41 kDa (gp41), ~20 kDa (Tr-Env-CT) and ~25 kDa (Ab LC) size were cut from the gel and transferred to a sterile microcentrifuge tube. The proteins were extracted from the gel, digested with trypsin, and the gp41 and Tr-Env-CT samples were deglycosylated with Peptide-N-Glycosidase F. The tryptic peptides were analyzed by a Q Exactive TM Hybrid Quadruple-Orbitrap TM mass-spectrometer coupled to a EASY nLC1000 (ThermoFisher Scientific) liquid chromatography. The m/z ratio of the detected peptides was compared to a database containing the human proteome and HIV-1 isolate BH10 protein sequences, for gp41 and Tr-Env-CT, and the Mus musculus proteome for Ab LC. Table S1. Proviral constructs expressing different variants of gp41.

Proviral pCHIV WT
Non-infectious plasmid expressing all HIV-1 NL4-3 proteins except Nef, but cannot replicate because of the lack of the viral long-terminal repeat sequences [6] .