Subnanometer structures of HIV-1 envelope trimers on aldrithiol-2-inactivated virus particles.

Cell lines and viruses HIV-1

BaL virus was produced by the HIV-1 BAL /SUPT1-CCR5 cell line. The cell line ID for it is “CLN204”. Its derivation was described previously 51 and the virus that it produces is dual tropic. HIV-1 BaL CL.1 virus was produced by the HIV-1 BAL /A66-R5 CL.1 limiting dilution clone [CLN445]. It was derived by infecting A66-R5 cells 52 with a HIV-1 BAL infectious stock obtained from the NIH AIDS Reagent Program (catalog # 510 – lot 100219) followed by preparation of limiting dilution clones. HIV-1 BaL CL.1 virus is R5 tropic.

Virus production and purification

Up to 30 L batches of cell culture propagated in roller bottles were serially filtered using 5.0 μm capsule filters to remove the cells followed by filtration using either 0.45 or 0.50 μm capsule filters to remove a fraction of the contaminating microvesicles 53 . The filtrate was treated with a final concentration 1mM Aldrithol-2 (AT-2) to inactivate the virus 54 , 55 and the virus was purified by continuous flow sucrose density gradient ultracentrifugation 51 . HIV-1 BAL virus lot P4311 and HIV-1 BAL CL.1 virus lot P4338 were imaged by cryoET.

CryoET sample preparation AT-2 treated BaL strain

HIV-1 virus 56 was incubated for 30 minutes at room temperature with ligands 10–1074 and 3BNC117, or sCD4 D1D2 and 17b. 6 nm colloidal gold solution was added to the mixture and 4 μl of them were placed onto freshly glow discharged holey carbon grids for 1 minute. Grids were blotted with filter paper from the back, and rapidly frozen in liquid ethane using a homemade gravity-driven plunger apparatus.

CryoET data collection

The frozen-hydrated grids were imaged at −170 °C on a cryo-electron microscope (Titan Krios, Thermo Fisher Scientific), equipped with a field emission gun, an energy filter, Volta phase plate, and a direct detection device (K2 Summit, Gatan). Volta phase plate enhances the contrast of the tomogram, helping a better visualization of the Env on virus surface ( Fig. 1a and b ). The microscope was operated at 300 keV with a magnification of 64,000×, resulting in 2.245 Å/pixel at the specimen level. The tomographic package SerialEM 57 was utilized to collect 35 image stacks at a range of tilt angles from −51° to +51° with increments of 3° for each tilt series. The raw images were collected from single-axis tilt series at focus with accumulative dose of ~50 e − /Å 2 . For every single tilt series collection, the does-fractionated mode was used to generate 8–10 frames per projection image.

Image processing and tomogram reconstruction

Collected dose-fractionated data was subjected to the motion correction using Motioncorr2 58 to generate drift-corrected stack files, which were aligned using gold fiducial makers by IMOD 59 , 60 . The reconstructions were generated by TOMO3D 61 . In total, 431 tomograms (3,838×3,710 ×1,600) were generated for HIV-1 viruses. Sub-tomogram analysis HIV-1 BaL virus particles and then Env trimers were manually picked and extracted from the tomograms. In total, 59,716 Env trimers were used for sub-tomogram averaging by using I3 (0.9.9.3) 62 , 63 . First, 4 × 4 × 4 binned Sub-tomograms were subjected to “alignment by classification” method to generate initial low-resolution structure without using any initial model as reference 63 , 64 . Second, we used unbinned sub-tomograms for further refinement. We explored the possibility of asymmetry (C1), but the trimers stayed at low resolution. Higher resolution in situ structures were determined when C3 symmetry was imposed. We used Resmap 65 to estimate the local resolution of cryoET maps.

Modeling and visualization

To obtain models for cryoET density maps at different states, we referred to several crystal structures and single particle cryoEM structures to individually generate gp120 and gp41 subunit model of BaL strain. Swiss model workspace 66 was used to generate homology models with BaL sequence. We used the ligand-free crystal structure of BG505 SOSIP.664 gp120 and gp41 subunits (PDB 4ZMJ) for the ligand-free maps; the PGT151-bound JR-FL ΔCT(PDB 5FUU) ligand-free gp41 atomic model for the 10–1074/3BNC117 cryoET map, and CD4–17b/21c-8ANC195 (PDB 6CE3 and 6EDU) for the CD4–17b bound cryoET map. Atomic structures of Fabs and ligand including 10–1074, 3BNC117, sCD4, 17b were docked into cryoET maps through the function “Fit in map” in UCSF Chimera 67 , retaining the binding interface within the trimers and ligands. We also used Phenix package 68 to refine the models and to estimate the resolution of the cryoET maps by comparing with the fitted models. All the density maps are segmented in the UCSF Chimera 67 , and ChimeraX 69 was used for surface rendering and visualization of cryoET maps and models.

Preparation of dye-labeled HIV-1

Env trimer on the virus for smFRET The preparation of fluorescently labeled HIV-1 NL4–3 , HIV-1 JR-FL (with and without cytoplasmic tail), and HIV-1 BG505 virus Env was as previously described 70 , 71 . Two short enzymatic labeling peptides Q3 and A1 (Q3: GQQQLG and A1: GDSLDMLEWSLM) were inserted into variable loops V1 and V4 (V1V4) of Env gp120 subunit, respectively. These peptides allow Env to be site-specifically labeled with organic dyes through enzymatic reactions. HIV-1 NL4–3 virus carrying labeled Env was generated by transfecting 80% confluent HEK293 cells with a 40:1 ratio of replication-defective (RT-deleted) wild-type full-length HIV-1 NL4–3 plasmid to V1V4-tagged Env HIV-1 NL4–3 plasmid. HIV-1 BG505 virus Env was generated in a similar fashion in which plasmids encoding RT-deleted full-length HIV-1 Q23 BG505 (Q23 as the backbone) and V1V4-tagged Env HIV-1 Q23 BG505. HIV-1 JR-FL virus Env were produced by pseudo-typing a NL4–3 backbone (RT-deleted, Env-deleted) with JR-FL wild-type or cytoplastic tail-deleted (ΔCT) Env at a ratio of 40:1. Viruses were harvested 40 hours post-transfection from the culture supernatant, concentrated through a 15% sucrose cushion at 25,000 rpm for 2 hours, and then resuspended in 50 mM HEPES pH 7.5 buffer with 10 mM MgCl 2 and 10 mM CaCl 2 . Virus suspensions were then incubated with customized conjugated organic dyes (0.5 μM Cy3B(3S)-cadaverine and 0.5 μM LD650-CoA, Lumidyne Technologies), and enzymes (5 μM AcpS and 0.65 μM transglutaminase, Sigma Aldrich) at room temperature overnight. The next day, viruses were first incubated with PEG2000-biotin (0.02 mg/ml, Avanti Polar Lipids) at room temperature for 30 min and then purified by density gradient centrifugation to remove free dyes. smFRET imaging, data acquisition, and analysis All smFRET experiments were performed on a customized prism-based total internal reflection fluorescence (TIRF) microscope, and data were analyzed using the SPARTAN software package 72 . Briefly, fluorescently labeled viruses were immobilized on passivated streptavidin-coated quartz slides. A continuous-wave 532-nm laser was equipped to excite donor (Cy3) fluorophores.

Fluorescence signals of both donor

(Cy3) and acceptor (Cy5) were collected by a 60 × (1.27-NA) water-immersion objective (Nikon), spectrally split, and then simultaneously recorded on two synchronized ORCA-Flash4.0v2 sCMOS cameras (Hamamatsu) at a speed of 40 milliseconds per frame. Each recorded FRET movie consists of 2000 frames that reflect the conformational motions of HIV-1 Env in real time. Viruses were imaged in Tris (pH 7.4, 50 mM) NaCl (50 mM) buffer, which contains a cocktail of triplet-state quenchers as well as oxygen scavengers consisting of protocatechuic acid (2 mM, PCA) and protocatechuic 3,4-dioxygenase (8 nM, PCD). When indicated, fluorescently labeled virus were pretreated by AT-2, matching the conditions used for the virus preparations used for cryoET. Specifically, viruses were incubated without and with 1 mM AT-2 at 37 °C for one hour, followed by 18 hours incubation on ice before imaging. For measurements of the conformational effects of antibodies, viruses were pre-incubated with 50 μg ml −1 V3-glycan patch targeting bNAb 10–1074 (NIH AIDS reagent program) and 50 μg ml −1 CD4 binding site-targeting 3BNC117 (NIH AIDS reagent program) for 30 mins at room temperature and remained present during the following imaging. Real-time fluorescence intensities of both donor (I D ) and acceptor (I A ) fluorophores were then extracted from recorded FRET movies. FRET efficiency was calculated based on FRET= I A /(γI D +I A ), where γ is the correlation coefficient that corrects for the discrepancy between donor and acceptor in quantum yields and detection efficiencies. FRET time trajectories or FRET traces that pass strict criteria of sufficient signal-to-noise (S/N) ratio and negative cross-correlation between donor and acceptor intensity were then compiled into FRET histograms (probabilities vs. FRET values). FRET histograms were further fitted into a sum of three Gaussian distributions based upon the direct observation of state-to-state transitions in each of the FRET traces and the following idealization of those transitions by Hidden Markov Modeling. The relative occupancy of each conformational state was estimated as the area under each Gaussian curve, and the standard error was derived from thousands of individual data points associated with a number ( n ) of molecules in the FRET histogram. Reporting Summary is available Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data availability statement

In situ structures of HIV-1 Env were deposited to the EMDB with the following codes: ligand-free Env: EMD 21412, CD4–17b bound Env: EMD 21411, and 10–1074&3BNC117 bound Env: EMD 21413.

Reporting Summary statement

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.