Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM.

Protein Expression and Purification. Both untagged HIV-1 CA and HIV-1 CA- 6x His were produced in BL21 Escherichia coli cells. Cells were grown to OD 600 ∼0.8 before induction with 400 uM isopropyl β-d-1-thiogalactopyranoside at 30 °C for 6 h and then collected by centrifugation. Cells were resuspended in lysis buffer (for untagged CA, 25 mM Tris, pH 8.0, 2 mM phenylmethylsulfonyl fluoride (PMSF), 4 mM tris(2-carboxyethyl)phosphine (TCEP); for CA- 6x His, 50 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole, 2 mM PMSF, and 4 mM TCEP) and lysed by sonication. Insoluble material was collected by ultracentrifugation and discarded. Polyethylenimine was added to the cleared lysate to 0.3% and mixed at 4 °C for 10 min. Bulk protein was then precipitated from the lysate with 20% ammonium sulfate (incubated, stirring, at 4 °C for 40 min), and precipitated protein was collected by centrifugation. The protein pellet was resuspended in CA lysis buffer and then run through a HiPrep desalting column (GE Healthcare Life Sciences) with 50 mM Tris, pH 8.0, and 2 mM TCEP. Protein peak fractions were collected and combined. For purification of untagged CA and CA K25N , CA was isolated from the desalted crude protein via a tandem HiTrap Q HP-HiTrap SP HP anion–cation exchange column setup: Crude protein was applied to the columns (Q first, followed immediately by SP), and flow-through containing CA was collected. Residual protein was collected by washing the column once with 25 mM Tris, pH 8, 2 mM TCEP and then twice with 25 mM Tris, pH 8, 25 mM NaCl, and 2 mM TCEP. Eluate and washes were combined, and buffer exchanged into 25 mM Tris, pH 8 and 2 mM TCEP via gel filtration (Superdex 75 Increase 10/300; GE Healthcare Life Sciences). Protein peak fractions were combined and concentrated to 1 mM, then flash-frozen in liquid nitrogen, and stored at −80 °C. For purification of CA- 6x His, NaCl and imidazole were added to the desalted crude protein to final concentrations of 300 mM and 10 mM, respectively. The solution was then incubated with NiNTA agarose resin (Qiagen) for 1 hr at 4°C, rotating. The resin was washed 1x in batch with 20 mL CA-His lysis buffer, then transferred to a gravity column, and washed 10x with 1 mL CA-His wash buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 30 mM imidazole, and 2 mM TCEP). CA- 6x His was then eluted in 1 mL fractions with CA-His elution buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 300 mM imidazole, and 2 mM TCEP). CA- 6x His was further purified by gel filtration (Superdex 75 Increase 10/300; GE Healthcare Life Sciences) in CA-His gel filtration buffer (25 mM Tris, pH 8.0, and 2 mM TCEP). Protein peak fractions were combined and concentrated to 500 uM, then flash-frozen in liquid nitrogen, and stored at −80 °C. In Vitro CA CLP Assembly. CLP assembly reactions were buffered with 50 mM MES, pH 6.2, and contained 500 uM purified untagged HIV-1 CA, 2.5 mM IP 6 (Tokyo Chemical Company), and 1 mM TCEP. CA was first warmed at 37 °C for 5 min, then combined with IP 6 , and incubated at 37 °C for 15 min. Assemblies were stored on ice or at 4 °C until grid preparation. Templated CA lattice assemblies were screened by negative stain transmission electron microscopy (TEM) (FEI Tecnai 12 BioTwin TEM or FEI Morgagni TEM; see Negative Stain EM in the Materials and Methods section) before cryogrid preparation. Liposome Preparation. Chloroform stock solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], nickel salt (DGS-NiNTA) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Nu-Chek Prep (Elysian, MN), and cholesterol stock solutions were prepared by standard gravimetric procedures to within 0.2% error. Stock solutions were mixed at an 85:10:5 DOPC:DGS-NiNTA:cholesterol ratio and then exchanged into 50 mM MES with 2 mM TCEP (pH 6.2 or 7.4) via rapid solvent exchange to a final total lipid concentration of 13 mM. SUVs were prepared by sonication in a room temperature water bath for 30 min (Sonblaster Series 200, Narda Ultrasonics Corporation) and then filtered through a desalting spin column (Thermo Fisher Scientific). LUVs were prepared by extrusion through a 100-nm polycarbonate filter using a miniextruder (Avanti Polar Lipids) 25 times at room temperature. CA Lattice Templating on Liposomes. Purified HIV-1 CA- 6x His was combined with liposomes and incubated at 37 °C for 5 min. 10 mM IP 6 (Tokyo Chemical Company) in 50 mM MES (pH 6.2 or 7.4) was added to a final concentration of 1 mM, and the assembly reaction was incubated at 37 °C for 15 min. SUV assembly reactions (pH 6.2 or 7.4) contained 330 µM CA- 6x His and 5.9 µM SUVs; LUV assembly reactions (pH 7.4) contained 190 mM CA- 6x His and 3.7 mM LUVs. If applicable, dNTPs (equimolar mixture of dATP, dCTP, dGTP, and dTTP; Cytiva) were added to a final concentration of 2 mM in place of IP 6 . For preparation of GS-6207-bound lattice, GS-6207 (Gilead Sciences) was added to a final concentration of 330 µM 5 min after IP 6 addition, and the reaction contained 125 mM NaCl and was buffered with 50 mM HEPES (pH 7.4) rather than MES. Templated lattice assemblies were screened by negative stain TEM (FEI Tecnai 12 BioTwin TEM; see Negative Stain EM in the Materials and Methods section) before grid preparation. Virus Production and Infectivity Assay. HIV-1 ΔEnv consisted of NL4-3-derived proviral vector with a 5′ cytomegalovirus-driven green fluorescent protein and defective for Vif, Vpr, Nef, and Env (kindly provided by Vineet Kewal-Ramani, National Cancer Institute-Frederick). CA mutations were made using the In-Fusion Cloning System (Takara Bio) using either custom Gene Blocks (Integrated DNA Technologies) (R18A, R18L, R18K, K25A, K25E, K25R, K25N, and K25N-N21K) or PCR site-directed mutagenesis (PR-D25A). All plasmids were verified by sequencing. The plasmid for vesicular stomatitis virus glycoprotein (VSV-g, NIH AIDS Reagent Program) has been previously described ( 52 ). HIV-1 VLPs were produced by Lipofectamine 3000 (Thermo Fisher Scientific) transfection of 293FT cells (purchased from Invitrogen; cells maintained as previously described in ref. 27 ) at ~50 to 60% confluence with 900 ng of the proviral vector and 100 ng of VSV-g. Media containing VLPs (“viral media”) were collected 2 d posttransfection. Viral media were then frozen at −80 °C for a minimum of 1 h to lyse cells, briefly thawed in a 37 °C water bath, and then precleared by centrifugation at 3,000 × g for 5 min. The supernatant was collected and added to fresh HEK293FT cells (6-well format) at low multiplicity of infection to prevent infection saturation. Infected cells were collected, washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 ), and treated with 10 mM TrypLE Express Enzyme (Gibco/Thermo Fisher Scientific). Cells were then resuspended in PBS, and 10% paraformaldehyde was added to a final concentration of 5%. After 10- to 20-min incubation at room temperature, the cells were collected by centrifugation at 500 × g for 5 min and resuspended in 500 μL PBS. Cells were assayed for fluorescence using an Attune Flow Cytometer with proprietary Attune collection and analysis software (Thermo Fisher Scientific).

Cell lysis and preparation of released

VLPs were carried out as previously described ( 27 ). VLPs were concentrated over 500 μL of 20% sucrose in PBS at 50,000 × g for 45 min (Beckman Coulter Optima TLX). Infections were assessed by western blot using the following antibodies: RbαHIV-p24 (provided by the HIV Reagent Program), AlexaFluor680-conjugated GtαRb-IgG (Life Technologies, A21076), and AlexaFluor790-conjugated MsαGAPDH (Santa Cruz Biotech, sc-365062). Blots were imaged using an Odyssey Imaging System (Lambda Instruments Corporation), and band densities were measured using Image Studio Lite software (Li-COR). Raw band densities were imported into Microsoft Excel, and infections were normalized to percentage of infections for wild-type virus, followed by normalization to a GAPDH loading control. Normalized data were then exported to CSV format for ANOVA and graph generation in RStudio ( 53 , 54 ). Negative Stain EM of CLPs. First, 4 µL sample was applied to a glow-discharged grid (Electron Microscopy Sciences Formvar/Carbon 200 Mesh) for 30 s. Then, the grid was washed with 100 μM IP 6 in 50 mM MES (pH 6.2 or 7.4) for ~5 s, stained with 2% uranyl acetate for 2 min, and blotted dry. Samples were imaged at 120 kV (FEI Tecnai 12 BioTwin TEM) or 100 kV (FEI Morgagni TEM). Cryo-ET and SPA Cryo-EM Sample Preparation. CA CLPs (3 to 4 μL sample) were applied to a glow-discharged grid (Protochips 2/2-3Cu C-Flat grids; PELCO easiGlow glow discharger), then blotted for 2 to 3 s, and plunge-frozen in liquid ethane. Plunging was performed at 90 to 100% humidity via automatic backside blotting (via Leica Microsystems Cryo-GP 2 plunge freezer) or dual-side blotting (Mark IV FEI/Thermo Fisher Scientific Vitrobot). CLP samples for cryo-ET contained BSA-coated 10-nm colloidal gold at a CLP:gold bead ratio of ~8:1. Liposome-templated CA (3.5 μL sample) was applied to a glow-discharged grid (QUANTIFOIL 300 mesh Au 1.2/1.3) and then manually wicked away from the edge of the grid with a Kimwipe (Kimberly-Clark). An additional 3.5 μL sample was applied, then automatically dual-side blotted for 3 s, and plunge-frozen in liquid ethane at 95% humidity/4 to 6 °C on a Mark IV FEI/Thermo Fisher Scientific Vitrobot. Cryo-ET and SPA Cryo-EM Data Collection. Cryo-ET samples were imaged at 300 kV on a Titan Krios TEM (Thermo Fisher Scientific) equipped with a K2-XP direct detector (Gatan, Inc.) and a BioQuantum postcolumn energy filter (Gatan Inc.) with a slit width of 20 eV. Imaging was done at a nominal magnification of 105,000× with a physical pixel size of 1.379 Å/pixel. Using SerialEM ( 55 ), dose-symmetric tilt series ( 56 ) were collected with a tilt angle range between −60° and 60°, 3° increment, and dose fractionation of each tilt image into 10 frames. SPA samples were imaged at 200 kV on a Talos Arctica TEM (Thermo Fisher Scientific) equipped with a K3 direct detector (Gatan, Inc.) and a BioQuantum energy filter (Gatan Inc.) with a slit width of 20 eV. Imaging was done at a nominal magnification of 63,000× in superresolution mode with a physical pixel size of 1.31 Å/pixel. Using SerialEM ( 55 ), a total of 50 frames were captured as movies. See SI Appendix , Table S1 . Cryo-ET and SPA Cryo-EM Data Processing. See SI Appendix , Table S1 . Cryo-ET and STA were performed essentially as previously described ( 5 , 57 ). Full details are provided in SI Appendix . SPA image processing was done in RELION 4.0 ( 58 ) [maintained by SBGrid ( 59 )] and CryoSPARC ( 60 ), and motion correction and CTF estimation were carried out using MOTIONCOR2 ( 61 ) and GCTF ( 62 ). The initial map used for subsequent alignments was generated by aligning CLP particles against EMDB-3465 ( 5 ) low pass filtered to 40 Å. Structures were determined using the pipeline described in SI Appendix , Fig. S2 .

Atomic Model

Building and Refinement. An initial reference model was prepared in UCSF Chimera ( 63 ) by docking the crystal structure of full-length hexameric CA (PDB 4XFX) into the cryo-EM map of SUV-templated CA lattice prepared at pH 7.4 with IP 6 and then refined using the Real Space Refinement tool in Phenix ( 64 ). The model was adjusted using the ISOLDE UCSF ChimeraX plugin ( 65 , 66 ), followed by iterative rounds of manual adjustments in Coot ( 67 ) and Real Space Refinements in Phenix ( 64 ). The resulting model was used as an initial model for all other modeled structures, which were also prepared as described above. Model quality was evaluated using MolProbity ( 68 ). Cryo-EM maps have been deposited into the EMDB, and atomic models have been deposited into the PDB. See SI Appendix , Table S1 . When preparing figures containing atomic models, IP 6 and GS-6207 were docked according to best cross-correlation with the cryo-EM density and are not included in the atomic models. RMSD measurements for structure comparisons were performed using the MatchMaker tool in ChimeraX ( 66 ).

Supplementary Material Appendix 01 (PDF) Click here for additional data file.

Data, Materials, and Software Availability Cryo-EM maps have been deposited into the Electron Microscopy Data Bank: EMD-16698 (CLP pentamer STA) ( 69 ), EMD-16699 (CLP hexamer STA) ( 70 ), EMD-29772 (lattice from CLPs) ( 71 ), EMD-29773 (templated lattice prepared with IP 6 at pH 6.2) ( 72 ), EMD-29774 (templated lattice prepared with IP 6 at pH 7.4) ( 73 ), EMD-29775 (templated lattice prepared with dNTPs at pH 7.4) ( 74 ), EMD-29776 (templated lattice prepared with IP 6 at pH 7.4; bound to GS-6207/lenacapavir) ( 75 ), and EMD-29777 (templated lattice prepared in the absence of polyanion) ( 76 ). Atomic coordinates have been deposited into the Protein Data Bank: PDB 8G6K (lattice from CLPs) ( 77 ), 8G6L (templated lattice prepared with IP 6 at pH 6.2) ( 78 ), 8G6M (templated lattice prepared with IP 6 at pH 7.4) ( 79 ), 8G6N (templated lattice prepared with dNTPs at pH 7.4) ( 80 ), and 8G6O (templated lattice prepared with IP 6 at pH 7.4; bound to GS-6207/lenacapavir) ( 81 ). All other data are included in the main article and/or in SI Appendix .