Evasion of cGAS and TRIM5 defines pandemic HIV

Cells and reagents

HEK293T and U87 cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Labtech) and 100 U ml−1 penicillin plus 100 μg ml−1 streptomycin (Pen/Strep; Gibco). THP-1-IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously62. THP-1 dual control and cGAS−/− cells were obtained from Invivogen. THP-1 IFIT1 cells were maintained in RPMI medium (Gibco) supplemented with 10% FBS and Pen/Strep. THP-1 dual cells were maintained in RPMI (Gibco) supplemented with 10% FBS, Pen/Strep, 25 mM HEPES (Sigma), 10 µg ml−1 of blasticidin (Invivogen) and 100 μg ml−1 of Zeocin (Invivogen). GHOST cells stably expressing CD4, CCR5, CXCR4 and the green fluorescent protein (GFP) reporter gene under the control of the HIV-2 long terminal repeat, were maintained in DMEM supplemented with 10% FBS, and antibiotics, G418 (500 μg ml−1) (Thermo Fisher), hygromycin (100 μg ml−1)(Invitrogen) and puromycin (1 μg ml−1) (Millipore). Lipopolysaccharide, IFNβ, IL-1β and poly I:C were obtained from Peprotech. Herring testes (HT) DNA was obtained from Sigma. For stimulation of cells by transfection, transfection mixes were prepared using lipofectamine 2000 (Invitrogen) in Optimem (Thermo Fisher). HT DNA and poly I:C concentration used are stated on each figure.

Isolation of primary MDM

Primary MDM were prepared from fresh blood from healthy volunteers. The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Lymphoprep (Stemcell Technologies), washed three times (PBS) and plated to select for adherent cells. Non-adherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10% heat-inactivated pooled human serum (Sigma) and 100 ng ml−1 macrophage colony stimulating factor (R&D systems). For replication experiments with full-length viruses, the medium was then refreshed after 3 d (RPMI 1640 with 10% human serum), removing any remaining non-adherent cells. After 6 d, media were replenished with RPMI containing 5% type AB human serum (Sigma-Aldrich). For single-round experiments with VSV-G-pseudotyped viruses, cells were washed (PBS) on day 3 of differentiation and the medium changed to RPMI supplemented with 10% heat-inactivated FBS. MDM were then infected 3–4 d later. Replicate experiments were performed with cells derived from different donors.

Editing of cells by CRISPR/Cas9

Lentiviral particles to generate CRISPR/Cas9-edited cell lines were produced by transfecting 10 cm dishes of HEK293T cells with 1.5 μg of pLentiCRISPRv2 encoding gene-specific guide RNAs (Addgene plasmid 52961), 1 μg of p8.91 packaging plasmid40 and 1 μg of VSV-G glycoprotein-expressing plasmid pMDG (Genscript) using Fugene-6 transfection reagent (Promega). Virus supernatants were collected at 48 and 72 h post transfection, pooled and used to transduce THP-1 IFIT1 cells by spinoculation (1,000 × g, 1 h, room temperature). Transduced cells were selected using puromycin (1 μg ml−1, Merck Millipore) and single clones isolated by limiting dilution in 96-well plates. Clones were screened for successful gene knock out by luciferase assay after targeted protein stimulation and immunoblotting.

gRNA sequences:

MAVS: CAGGGAACCGGGACACCCTC

Non-targeting control: ACGGAGGCTAAGCGTCGCAA

Virus plasmids

The NL4.3 (Ba-L Env), YU2 (ref. 31) and O-group molecular clones, RBF206 and BCF120, and HIV-2 molecular clones pJK7312S3 and pST63 have all been described. HIV-2 ROD10 was obtained from the National Institute of Biological Standards and Controls64. The CA chimera molecular clone was generated by overlap PCR, replacing CA residues 1–204 of NL4.3 with the equivalent residues from MVP5180 or HIV-2 ROD10. VSV-G-pseudotyped GFP-encoding vectors include HIV-1 M LAI ΔEnv.GFP (LAI strain39) with the Nef coding region replaced by GFP, HIV-1(M) R9 packaging vector (p8.91) and minimum genome-expressing GFP (CSGW)41. HIV-2 ROD GFP has been described65. HIV-1(O) packaging plasmid to make HIV-1(O) GFP was generated by replacing Gag-Pro residues between Not1–Bcl1 in p8.91 with the equivalent residues from MVP5180 (ref. 66). Q50Y and 120R mutations were generated by site directed mutagenesis of p8.91 using PfuTurbo (Agilent Technologies). For TREX1 over-expression, we used MLV-based gammaretroviral expression vector EXN67, where TREX1 coding sequence was cloned from a plasmid kindly provided by Nan Yan between BamHI and XhoI sites. For TRIM5 depletion with short hairpin (shRNA), we expressed shRNA using SIREN-RetroQ (Clontech) gammaretroviral vector containing shRNA sequence targeting human TRIM5 (ref. 26) or scramble Ctrl29 as described.

Production of virus in HEK293T cells

Replication competent HIV were produced by transfection of HEK293T cells in T150 flasks using Fugene-6 transfection reagent (Promega). Briefly, just-subconfluent T150 flasks were transfected with 8.75 μg of vector and 30 µl Fugene-6 in 500 µl Optimem (Thermo Fisher). Virus supernatants were collected at 48, 72 and 96 h post transfection. Virus suspensions were filtered, subjected to ultracentrifugation through a 20% sucrose buffer and resuspended in RPMI 1640 with 5% human serum for subsequent replication experiments in MDM. For VSV-G-pseudotyped GFP-expressing virus, each T150 flask was transfected with 2.5 μg of VSV-G glycoprotein-expressing plasmid pMDG (Genscript) and 6.25 μg of pLAIΔEnv GFP or 2.5 μg packaging plasmid (p8.91, MVP or HIV-2-pack) and 3.75 μg of GFP-encoding genome plasmid (CSGW or HIV-2 GFP) using 30 µl Fugene-6 in 500 µl Optimem. In the case of VLP without genome, the cells in T150 flasks were transfected only with 2.5 μg of VSV-G glycoprotein-expressing plasmid pMDG and 5 μg packaging plasmid (p8.91, MVP or HIV-2). Virus supernatants were collected at 48 and 72 h post transfection, pooled, DNase treated (2 h at 37 °C, DNaseI, Sigma) and subjected to ultracentrifugation over a 20% sucrose cushion. Viral particles were finally resuspended in RPMI supplemented with 10% FBS. Lentiviral particles to generate TREX-expressing vector or TRIM5 shRNA vector were produced by transfecting 10 cm dishes of HEK293T cells with 1.5 μg TREX EXN vector or TRIM5-targeting SIREN-RetroQ, 1 μg of packaging plasmid CMVintron, and 1 μg of VSV-G glycoprotein-expressing plasmid pMDG using Fugene-6 transfection reagent. Virus supernatants were collected at 48 and 72 h post transfection, pooled and stored at −80 °C.

Virus quantification and RT products

Full-length HIV clones were quantified by RT enzyme-linked immunosorbent assay (ELISA) (Roche). Reverse transcriptase activity of virus preparations was quantified by qPCR using a SYBR Green-based product-enhanced RT (SG-PERT) assay as previously described68. For viral genome copy measurements, RNA was extracted from 2 μl sucrose-purified virus using the RNeasy mini kit (QIAGEN). The RNA was then treated with TURBO DNase (Thermo Fisher) and subjected to reverse transcription using Superscript III reverse transcriptase and random hexamers (Invitrogen). Genome copies were then measured by Taqman qPCR using primers against GFP69 (see below).

For RT product measurements, DNA was extracted from 5 × 105 infected cells using the DNeasy blood and tissue kit (QIAGEN). DNA concentration was quantified using a Nanodrop for normalization. RT products were quantified by Taqman qPCR using TaqMan gene expression master mix (Thermo Fisher) and primers and probe specific to GFP. A dilution series of plasmid encoding GFP was measured in parallel to generate a standard curve to calculate the number of GFP copies.

Primers:

GFP fwd: 5′- CAACAGCCACAACGTCTATATCAT -3′

GFP rev: 5′- ATGTTGTGGCGGATCTTGAAG -3′

GFP probe: 5′- FAM-CCGACAAGCAGAAGAACGGCATCAA-TAMRA -3′

Infection assays

To measure viral replication, MDM were infected with 100 pg RT of full-length viruses, measured by RT ELISA (Roche) per well (multiplicity of infection (MOI) = 0.2) in 48-well plates and subsequently fixed and stained using mixed CA antibodies EVA365 and EVA366 (National Institute of Biological Standards AIDS Reagents) at 1/50, with goat anti-mouse immunoglobulin (Ig) antibody conjugated to β-galactosidase (926-32210, Southern Biotechnology Associates) at 1/15,000, and counted31. Anti-IFN-α/β receptor (PBL Interferon Source) or control IgG2A antibody (R&D systems) were added at 1 μg ml−1 for 2 h before infection and supplemented every 4 d. Single-round infection by VSV-G-pseudotyped viruses was performed in 48-well plates using equal viral doses (1 × 109 genome copies). Viral infection was measured 48 h later by enumeration of GFP-positive cells by flow cytometry. For RNA extraction and subsequent qPCR analysis, cells were infected in 24-well plates.

GHOST cells were infected with full-length viruses as previously described31, measured by RT ELISA per well in 48-well plates. Cells were fixed at the indicated times post infection and GFP+ cells measured by flow cytometry.

Monocytic THP-1 cells were infected at a density of 2 × 105 cells per ml in 48-well plates in the presence of polybrene (8 μg ml−1, Sigma). Infection levels were assessed at 48 h post infection through enumeration of GFP-positive cells by flow cytometry. Input dose of virus was normalized either by RT activity (measured by SG-PERT) or genome copies (measured by qPCR) as indicated. THP-1 cells were treated with similar doses of VLP normalized by SG-PERT as indicated. After 24 h, cells were infected with equal amounts of genome copies (2 × 108) and infection levels were measured 48 h post infection through enumeration of GFP-positive cells by flow cytometry.

THP-1 cells stably expressing TREX were generated by transduction with the MLV-based gammaretroviral expression vector EXN and maintained under selection with G418 (500 μg ml−1).

IFNβ (100 ng ml−1) or IL-1β (10 ng ml−1) were added at different time points to THP-1 cells, which were then infected at an MOI of 0.3. Infection levels were measured after 48 h by flow cytometry.

Luciferase and secreted alkaline phosphatase reporter assays

Gaussia/Lucia luciferase activity was measured by transferring 10 μl supernatant to a white 96-well assay plate, injecting 50 μl per well of coelenterazine substrate (Nanolight Technologies, 2 μg ml−1) and analysing luminescence on a FLUOstar OPTIMA luminometer (Promega). Data were normalized to a mock-treated control to generate a fold induction. Secreted alkaline phosphatase was measured using QUANTI-Blue (Invivogen), using 20 µl of cell supernatant.

Quantitative RT-PCR

RNA was extracted from MDM or THP-1 cells using RNAeasy (QIAGEN). RNA (500 ng) was used to synthesize complementary DNA using Superscript III reverse transcriptase (Invitrogen). cDNA was diluted 1:5 in water and 2 μl was used as a template for real-time PCR using SYBR Green PCR master mix (Applied Biosystems) and QuantiStudio 5 real-time PCR machine (Applied Biosystems). Expression of each gene was normalized to an internal control (GAPDH) and values were then normalized to mock-treated control cells to yield a fold induction. Primers:

GAPDH: Fwd 5′-GGGAAACTGTGGCGTGAT-3′, Rev 5′-GGAGGAGTGGGTGTCGCTGTT-3′

CXCL10: Fwd 5′-TGGCATTCAAGGAGTACCTC-3′, Rev 5′-TTGTAGCAATGATCTCAACACG-3′

IFIT2: Fwd 5′-CAGCTGAGAATTGCACTGCAA-3′, Rev 5′-CGTAGGCTGCTCTCCAAGGA-3′

MxA: Fwd 5′-ATCCTGGGATTTTGGGGCTT-3′, Rev 5′-CCGCTTGTCGCTGGTGTCG-3′

CCL5: Fwd: 5′-CCCAGCAGTCGTCTTTGTCA-3′, Rev 5′- TCCCGAACCCATTTCTTCTCT-3′

IFIT1: Fwd: 5′- CCTCCTTGGGTTCGTCTACA-3′, Rev 5′-GGCTGATATCTGGGTGCCTA-3′

IL-8: Fwd: 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′, Rev 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′PTGS2: Fwd: 5′-CTGGCGCTCAGCCATACAG-3′, Rev 5′-CGCACTTATACTGGTCAAATCCC-3′

IL-1β: Fwd: 5′-ATGATGGCTTATTACAGTGGCAA-3′, Rev 5′-GTCGGAGATTCGTAGCTGGA-3′

SOD2: Fwd: 5′-GGAAGCCATCAAACGTGACTT-3′, Rev 5′-CCCGTTCCTTATTGAAACCAAGC-3′

cGAS: Fwd 5′GGGAGCCCTGCTGTAACACTTCTTAT-3′ Rev, 5′-TTTGCATGCTTGGGTACAAGGT-3′

TREX: Fwd 5′CGCATGGGCGTCAATGTTTT-3′ Rev, 5′-GCAGTGATGCTATCCACACAGAA-3′

TRIM5 expression levels were measured using TaqMan gene expression assay detecting TRIM5 (FAM dye-labelled, TaqMan probe Hs01552559_m1), or the housekeeping gene OAZ1 (FAM dye-labelled, primer-limited, TaqMan probe Hs00427923_m1).

ELISA

Cell supernatants were collected for ELISA at 48 h post infection/stimulation and stored at −20 °C. CXCL10 and IL-8 protein were measured using Duoset ELISA reagents (R&D Biosystems).

cGAS and TRIM5 depletion by RNAi

MDM (1 × 105) differentiated in macrophage colony stimulating factor for 4 d were transfected with 25 pmol of siRNA SMART pool against cGAS (L-015607-02-0005), TRIM5 (L-007100-00-0005) or non-targeting control (D-001810-10-05) (Dharmacon) using lipofectamine RNAiMAX transfection reagent (Invitrogen). Medium was replaced after 18 h with RPMI 1640 supplemented with 10% FCS and cells cultured for an additional 3 d before infection. THP-1 dual cells (5 × 105 ml−1) were transfected with 35 pmol of siRNA SMART pool against cGAS, TRIM5 or non-targeting control (Dharmacon) using lipofectamine RNAiMAX (Invitrogen). Medium was replaced after 18 h with RPMI 1640 supplemented with 10% FCS and cells were plated in 48-well plates and infected as indicated. To deplete TRIM5 in U87 cells, pSIREN-RetroQ expressing shRNA TRIM5 was transduced at an MOI 1 and shRNA-expressing cells selected with 10 μg ml−1 puromycin. TRIM5 and cGAS expression were quantified by qPCR normalized to OAZ1 and GAPDH, respectively, by the delta delta threshold cycle (ΔΔCt) method.

Immunoblotting

Cells were lysed in 50 mM Tris buffer (pH 8), 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 0.05% (v/v) NP40 supplemented with protease inhibitors (Roche), clarified by centrifugation (14,000 × g for 10 min), and the supernatants boiled (5 min in 6X Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue, 100 mM β-mercaptoethanol)) before separation on 12% polyacrylamide gel. Proteins were transferred to Hybond ECL membrane (Amersham Biosciences) using a semi-dry transfer (Biorad). Primary antibodies goat anti-MAVS Ab (Cell Signaling, 3993, 1:1,000 dilution) and goat anti-tubulin Ab (Abcam, ab6046, 1:20,000 dilution) were detected with IRDye 800CW goat anti-rabbit IgG (H + L) (LI-COR, 926-32211, 1:20,000) and membranes imaged with an Odyssey CLX infrared imager (LI-COR Biosciences) using Image Studio V5.2.

Single-molecule analysis

Single-particle traces of AF488-CypA-labelled capsids inside permeabilized virions immobilized on a coverslip were recorded by total internal reflection (TIRF) microscopy and analysed by step fitting to determine distributions of capsid lifetimes for WT and mutant HIV50.

Phylogenetics

A dataset of representative HIV and SIV sequences from the CA region of gag were downloaded from the Los Alamos HIV-1 sequence database and aligned manually. The phylogeny was estimated from the nucleotide sequences using RAxML v8 (ref. 70) with substitution model GTR+ Gamma and rooted consistent with phylogenies that include non-primate lentivirus outgroup taxa71. ChromaClade v1.1 was used to annotate taxon labels with residues found at capsid protein sites45. Note that chromaclade does not use statistical tests to assess viral evolution. Rather, it provides a simple way to visualize lineage-specific amino acid variation in a qualitative and intuitive way. In this study, our focus on positions CA50 and 120 was also influenced by the hexamer structures, which revealed that these positions have a role in the structural differences observed between viral capsid hexamers.

Protein production and purification

HIV-1(M) CA R120

Protein was expressed and purified as previously described for HIV-1(M) CA WT72. In brief, HIV-1(M) CA R120 was expressed in E. coli C41 OverExpress C41(DE3) (Lucigen) in 1 l 2YT media supplemented with 100 μg ml−1 ampicillin at 37 °C and 250 r.p.m. until optical density at 600 nm reached 0.5, followed by the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce expression overnight at 14 °C. Cells were lysed in 50 mM Tris-HCl, 40 mM NaCl and 20 mM β-mercaptoethanol (pH 4.5) using a cell disruptor, followed by removal of the insoluble fraction (30,000 × g for 20 min). Capsid protein was precipitated with 20% (w/v) ammonium sulfate and pelleted (30,000 × g for 20 min). Pellets were resuspended in refolding buffer (100 mM citric acid, 20 mM TRIS (pH 4.5)), followed by extensive dialysis against the same buffer and then 25 mM Tris-HCl (pH 8). The capsid was further purified with anion exchange chromatography (AEC) using a 5 ml Hi-TRAP Q column (Cytiva). AEC purification was performed using buffer A (25 mM Tris-HCl (pH 8)) and buffer B (25 mM Tris-HCl, 1 M NaCl (pH 8)). Lastly, size exclusion chromatography (SEC) was performed using a Superdex 16/600 75 pg column with 25 mM Tris-HCl and 40 mM NaCl (pH 8). The protein was further concentrated to 3.0 mg ml−1 in the size exclusion chromatography buffer for crystallization.

HIV-1(O), HIV-1(M) CA Q50Y, SIVmac, SIVcpzPtt

Hexameric CA proteins, stabilized by engineered inter-subunit disulfide bonds, were produced by assembly of recombinant CA containing four amino acid substitutions73: HIV-1(O-group) (A14C, E45C, W185A, M186A); HIV-1(M-group) (A14C, E45C, Q50Y, W184A, M185A); SIVmac (P13C, E44C, W182A, M183A); and SIVcpz (P14C, E45C, W184A, M185A). Expression was performed in E. coli (C41) by mid-log induction with 1 mM IPTG14 overnight at 14 °C. Collected cells were lysed in 50 mM Tris (pH 8.0), 150 mM NaCl and 20 mM β-mercaptoethanol by sonication. Clarified lysates were treated with 20% (w/v) ammonium sulfate and the precipitate resuspended in 100 mM citric acid (pH 4.5) and 20 mM β-mercaptoethanol, and dialysed against the same to remove the ammonium sulfate. Redissolved protein was subjected to assembly by three dialysis steps: (1) 1 M NaCl, 50 mM Tris (pH 8.0), 20 mM β-mercaptoethanol; (2) 1 M NaCl, 50 mM Tris (pH 8.0); and (3) 20 mM Tris (pH 8.0), 40 mM NaCl. Purified hexamers were isolated by size exclusion chromatography using a 16/600 Superdex 200 Prep Grade column on an ÄKTA Pure with 20 mM Tris and 40 mM NaCl.

Crystallization, structure solution and analysis

HIV-1(M) CA R120

Crystals were grown using the hanging-drop vapour-diffusion technique at 20 °C by mixing 1 μl protein with 1 or 2 ul precipitant containing 9.5–11% (w/v) PEG3350, 250–350 mM NaI and 100 mM sodium cacodylate (pH 6.5) as previously described74. Collected crystals were immersed in precipitant mixture with 20% (v/v) glycerol and cryo-cooled in liquid nitrogen. Diffraction data were collected from a single crystal at the PETRA III P13 beamline (EMBL Hamburg/DESY P13, Germany). The dataset was indexed, processed and scaled using XDS vJan31,2020 (ref. 75). The HIV-1(M) CA R120 crystal belonged to the P6 space group with a solvent content of 48.5% corresponding to one molecule per asymmetric unit. The structure was determined by molecular replacement using Phenix Phaser v2.8.3 (ref. 76) and a previously determined HIV-1(M) CA structure (PDB ID:4XFX) as search model. Model building was performed using COOT v0.8.9.2 (ref. 77). Refinement was performed using REFMAC v5.8. Overview of refinement procedures was within REFMAC5: utilizing data from different sources78 using a TLS/maximum-likelihood protocol. The model converged to a final Rwork/Rfree of 0.242/0.277 at a resolution of 2.30 Å. The HIV-1(M) CA R120 model covers the HIV-1(M) CA amino acid sequence 1–222 and contains in addition 2 iodine, 4 chlorine ions and 14 water molecules. Figures were rendered using PyMOL (version 2.5.0.a0, Schrodinger).

HIV-1(O), HIV-1(M) CA Q50Y, HIV-1(M) CA Q50Y/R120, SIVmac, SIVcpz

HIV-1(O-group) hexamer crystals were grown using hanging-drop vapour-diffusion with 2 μl protein (40 mg ml−1) +2 μl crystallant (10% (w/v) PEG 6000, 100 mM HEPES (pH 7.0), 100 mM glycine) suspended over 500 μl undiluted crystallant. Crystals were cryoprotected with the gradual addition of glucose (solid) to 40% (w/v). HIV-1(M-group, Q50Y) hexamer crystals were grown using hanging-drop vapour-diffusion with 2 μl protein (13 mg ml−1) +2 μl crystallant (19% (v/v) PEG 550MME, 100 mM Tris (pH 8.0), 150 mM KSCN, 10 mM ATP, 3% (v/v) 3,5-hexanediol) suspended over 500 μl undiluted crystallant and cryoprotected in 20% (v/v) 2-methyl-2,4-pentanediol (MPD). HIV-1(M-group, Q50Y/R120) hexamer crystals were grouped using sitting-drop vapour-diffusion with 1 μl protein (12 mg ml−1) +1 μl crystallant (20% PEG 550MME, 0.1 M Tris (pH 8.0), 0.15 M KSCN, 10 mM ATP, 3% ethanol) suspended over 80 μl crystallant and cryoprotected in 20% (v/v) MPD. SIVmac hexamer crystals were grown using sitting-drop vapour-diffusion with 200 nl protein (12 mg ml−1) +200 nl crystallant (10% (w/v) PEG 6000, 5% (w/v) MPD, 100 mM HEPES (pH 7.5)) suspended over 80 μl crystallant and cryoprotected in 20% (v/v) MPD. SIVcpz hexamer crystals were grown using sitting-drop vapour-diffusion using 200 nl protein (12 mg ml−1) +200 nl crystallant (4.5% PEG 550MME, 0.15 M KSCN, 0.1 M Tris (pH 9.0), 4% 2,5-hexanediol) suspended over 80 μl crystallant and cryoprotected in 20% (v/v) MPD. Diffraction data were collected at 100 K on Diamond Light Source beamlines I02 (HIV-1(O), HIV-1(M), Q50Y) and I04-1 (SIVmac, SIVcpz) or in-house (M-group Q50Y/R120) on a Rigaku FR-E Superbright rotating anode source equipped with an MAR345 image plate detector. Data were reduced using IMOSFLM v7.4 (ref. 79) or XDS v0.6.5.2 (ref. 75), and scaled and merged using AIMLESS v0.7.4 (ref. 80). Structures were solved by molecular replacement using PHASER v2.8.3 (ref. 81) and search model based on the original cross-linked HIV-1(M) hexamer, PDB:3H47 (ref. 73). Structures were refined using REFMAC5 v5.8 (ref. 82) or phenix.refine v1.17.1.3660 (ref. 83). Between rounds of refinement, models were manually checked and corrected against the corresponding electron-density maps in COOT84. The quality of the model was regularly checked for steric clashes, incorrect stereochemistry and rotamer outliers using MOLPROBITY v4.02b-467Xtriage85.

Position-specific scoring matrices (PSSMs)

Sequence alignments for the capsids of HIV-1(M), HIV-1(O), HIV-2, SIVcpzPtt_and SIVcpzPts_ were either obtained as pre-made alignments from the LANL HIV Database, or directly from NCBI Virus followed by multiple sequence alignment using Clustal Omega. All alignments were manually adjusted, and sequences with larger insertions, deletions and/or poor sequence coverage were excluded. PSSMs (Supplementary Table 1) were generated using an R-script provided by Julian Villabona Arenas.

Statistical analysis

We have included the number of replicates (equal to the number of different donors), statistical tests and significance criteria in figure legends and in the main text. Statistical analysis was performed in GraphPad Prism. The following P values were considered significant: ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. Data collection and refinement statistics of the protein structures solved in this paper can be found in Supplementary Table 2.

Reporting summary

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

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