Evasion of cGAS and TRIM5 defines pandemic HIV

Abstract

Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.

Fig. 1. HIV activation of innate immune responses in macrophages.

a–c, Replication of HIV-1(M) (a), HIV-2 (b) or HIV-1(O) (c) isolates in human MDM in the presence of interferon α/β receptor (IFNα/β-R) or control antibody (CAb). Two-way ANOVA vs CAb, ROD10 P = 0.0001, pSTbx P = 0.0001, ps7312s P = 0,0001, RBF206 P = 0.033. d, Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. e, Secreted IL-8 and CXCL10 from infections in d measured by ELISA 48 h post infection. f, GAPDH-normalized mRNA levels in infections from d expressed as fold induction over untreated MDM 24 h post infection or after HT-DNA transfection (1 ug ml⁻¹) or LPS stimulation (100 ng ml⁻¹). g, Infection of THP-1 cells with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. h, GAPDH-normalized mRNA levels from infections in g expressed as fold induction over untreated THP-1 cells 24 h post infection. Mean ± s.d., n = 3 donors (a–e) or independent experiments (f–h). Two-tailed unpaired t-test vs untreated MDM (d–f), paired t-test vs untreated THP-1 cells (g,h). *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Source data

Fig. 2. cGAS and TRIM5 detect non-pandemic HIV.

a, Example of GAPDH-normalized cGAS mRNA levels in MDM representing 3 independent donors transfected with cGAS-targeting siRNA (sicGAS), TRIM5-targeting siRNA (siTRIM5) or non-targeting control (siCtrl). b–e, GAPDH-normalized mRNA levels (left and middle), or secreted cytokine levels (right) (ELISA) (CXCL10, IL-8), expressed as fold induction over uninfected (mock) samples 24 h post infection (mRNA) or 48 h post infection (cytokine) from cells in a. f, GAPDH-normalized mRNA levels, expressed as fold induction over uninfected samples, in non-targeting CRISPR-treated cells (NT-KO) transfected with control siRNA (siCtrl) (NT-KO siCtrl), NT-KO siTRIM5, cGAS KO siCtrl or cGAS KO siTRIM5 THP-1 cells 24 h post infection or after HT-DNA (1 ug ml⁻¹) transfection. Mean ± s.d., n = 3 independent experiments and donors. Two-tailed unpaired t-test vs untreated MDM (b–e), paired t-test vs THP-1 Ctrl vector (f). *P < 0.05, **P < 0.01. Source data

Fig. 3. Genome-free HIV activates TRIM5, but not cGAS, to induce an antiviral state.

a, GAPDH-normalized mRNA levels expressed as fold induction over uninfected THP-1 cells 24 h post viral-like particles (VLP) treatment. b, HIV-1(M), HIV-2 and HIV-1(O) infection levels normalized to levels of infection without previous exposure to VLP. Mean ± s.d., n = 3 independent experiments. Two-tailed paired t-test vs untreated THP-1 (a). *P < 0.05, **P < 0.01. VSV-G-pseudotyped VLP were made without genome. Source data

Fig. 4. Pandemic-associated adaptation of HIV capsid at position 50.

a, Replication of HIV-1(M) NL4.3 (BalEnv) bearing HIV-2 ROD10 (top) or (O) MVP5180 (bottom) capsid in MDM in the presence of IFNα/β-R or CAb. Data show mean + s.d. n = 3 donors. Two-way ANOVA vs CAb, HIV-1(HIV-2 CA) P = 0.02, HIV-1(O CA) P = 0.002. b, HIV-1(M) CA hexamer highlighting β-hairpin (BHP) position in a closed (green) (PDB ID:5HGN) or open conformation (wheat) (PDB ID:5HGL). Lower panel details residues in hinge region that close BHP by coordinating water and increasing distance between His12 and Asp51. c, Maximum-likelihood phylogenetic tree of primate lentiviral capsid genes coloured by chromaclade to illustrate the residues equivalent to CA Q50 in HIV-1(M). d, SIVcpz CA hexamer (PDB ID:7T15) with the BHP position in a closed (green) conformation. Lower panel details residues in hinge region that close BHP by coordinating water and increasing distance between His12 and Asp51. e,f, HIV-1(O) (PDB ID: 7T12) and SIVmac (PDB ID:7T14) hexamers with open BHP (wheat) and key Gln at position 50 substituted for Tyr (Y), preventing water coordination and channel closure. Lower panels show the BHP hinge region in detail. g, A modelled hexamer built from the HIV-2 N-terminal CA domain (PDB ID:2 × 82) is shown for comparison with HIV-1(M) and (O). The HIV-2 BHP (wheat) is open. Note that HIV-2 position 49 is Tyr (Y). h, Crystal structure of HIV-1(M) Q50Y (PDB ID:7T13) highlighting BHP position in an open conformation (wheat). R18 is shown at the centre of the hexamers. Lower panels show the BHP hinge region in detail. i, WT and CA Q50Y capsid survival curves obtained from TIRF in vitro uncoating experiments. In the absence of IP6, HIV-1 Q50Y capsids are metastable and disassemble spontaneously with similar half-life to WT. Most capsids are stabilized in the presence of 100 μM IP6. Survival curves were generated from single-virion uncoating traces (N = 326 for WT, 326 for WT + 100 μM IP6, 326 for Q50Y, 326 for Q50Y + 100 μM IP6) from one representative uncoating experiment (see Extended Data Fig. 5 for additional Q50Y data). Source data

Fig. 5. Pandemic-associated adaptation of HIV capsid at position 120.

a, Maximum-likelihood phylogenetic tree of primate lentiviral capsid genes coloured to illustrate the residues equivalent to HIV-1(O) CA 120. Grey and (_) branch labels denote a gap in the alignment. b, Structures showing salt bridges in HIV-1(O) (PDB ID:7T12) (E98-R120), HIV-2 (PDB ID:2 × 82) (E96-R118), SIVmac (PDB ID:7T14) (E95-R117) and HIV-1(M) + R120 (PDB ID:7QDF) (E98-R120). The salt bridge is absent in WT HIV-1(M) CA (PDB ID:5HGN) and SIVcpz (PDB ID:7T15) because R120 is absent. Helix bearing R120 in HIV is coloured blue. Salt bridges are shown as dashed lines. CypA binding loop is coloured wheat. c, Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) or HIV-1(M) CA Q50Y 120R GFP measured at 48 h post infection by flow cytometry. Mean ± s.e.m. n = 3 donors. Source data

Fig. 6. Capsid mutations make pandemic HIV-1(M) sensitive to cGAS and TRIM5.

a, Replication of HIV-1(M) NL4.3 (BalEnv) WT or HIV-1(M) NL4.3 (BalEnv) bearing CA Q50Y + 120R in MDM in the presence of IFNα/β-R or CAb. b, GAPDH-normalized mRNA levels induced by HIV-1(M) CA Q50Y 120R, expressed as fold induction over uninfected samples in control siRNA-transfected (siCtrl) or cGAS siRNA-transfected (sicGAS) MDM 24 h post infection. c, GAPDH-normalized mRNA levels induced by HIV-1(M) CA Q50Y 120R expressed as fold induction over uninfected samples in control siRNA (siCtrl)- or TRIM5 siRNA (siTRIM5)-transfected MDM 24 h post infection. d, GAPDH-normalized mRNA levels induced by HIV-1(M) CA Q50Y + 120R expressed as fold induction over uninfected samples in non-targeting CRISPR-treated cells (NT-KO) transfected with control siRNA (siCtrl) (NT-KO siCtrl), NT-KO siTRIM5, cGAS KO siCtrl or cGAS KO siTRIM5 THP-1 cells measured 24 h post infection. e, GAPDH-normalized mRNA levels induced by HIV-1(M) Q50Y + 120R VLP (no genome) expressed as fold induction over uninfected THP-1 cells measured 24 h post infection. Mean ± s.d., n = 3 independent experiments or donors. Two-way ANOVA vs CAb (a), two-tailed unpaired t-test vs siCtrl (b,c), paired t-test vs NT-KO siCtrl THP-1 cells (d,e). *P < 0.05, **P < 0.01. Source data

Extended Data Fig. 1.

a. Infection of MDM with HIV, measured at 48 h by counting Gag positive cells, in the presence of anti-interferon α/β receptor (IFNα/β-R), or control, antibody (cAb). b. Replication of HIV-1(M), HIV-2 or HIV-1(O) isolates in permissive GHOST cells measuring induced GFP expression by flow. Representative experiment of 2 independent replicates. c, d. Activation of (c) IRF-luciferase reporter or (d) NF-kB secreted alkaline phosphatase reporter 48 h after infection by equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 or HIV-1(O) -GFP. e, f. Measurement of VSV-G pseudotyped HIV-1(M), HIV-1(O) and HIV-2 DNA synthesis (GFP primers) during a 20 h time course in THP-1 cells. g. Infection measured at 48 hours in wells parallel to (e) by flow. h. Viral DNA (GFP) copy number at 20 h post-infection per infected cell using data from (e-f). Mean + /− SD, N = 3 donors (MDM) or independent experiments (THP-1 c,d). N = 4 independent experiments ThP1 e-h. Source data

Extended Data Fig. 2

a. GAPDH-normalised TRIM5 mRNA levels, measured after transfection of THP-1 treated with non-targeting CRISPR (NT-KO) or cGAS CRISPR (cGAS-KO) and transfected with TRIM5 targeting siRNA (siTRIM5) or non-targeting control siRNA (siCtrl). N = 3 independent experiments. b. Anti-MAVS or tubulin western blot of non-targeting KO cells (NT-KO) or MAVS KO cells. c. % infection of HIV-1(M), HIV-2 and HIV-1(O) in NT-KO or MAVS KO cells. d. IFIT1 reporter activation after viral infection normalised against mock infected. e. GAPDH-normalised mRNA levels expressed as fold induction over mock-treated non-targeting KO control (NT-KO) or MAVS KO THP-1 cells 24 h post-infection or after poly I:C transfection (500 ng/mL). Mean + /− SD, n = 2 independent experiments. Source data

Extended Data Fig. 3

a. A representative example of GAPDH-normalised TREX1 mRNA levels measured after transduction of THP-1 cells with empty (Ctrl) MLV vector or TREX1 expression vector. b, c. GAPDH-normalised ISG mRNA levels, expressed as fold induction over uninfected, in control vector (Ctrl) or TREX1-expressing THP-1 cells with HIV-2 and HIV-1(M) or HIV-1(O) with d, e. infection levels in parallel wells measured 48 hpi. Mean + /− SD, n = 3 independent experiments. b,d two-tailed paired t-test vs THP-1 Ctrl vector. *p < 0.05. Source data

Extended Data Fig. 4

a. NF-kB - secreted alkaline phosphatase reporter 24 or 72 hours after treatment with increasing doses of viral-like particles (VLP, made with packaging plasmid and VSV-G but no genome). N = 2 independent experiments. b. Quantification of IL-1β in supernatants from MDM mock infected or infected with HIV-1(M), HIV-2 or HIV-1(O). N = 4 donors. c. IRF-reporter activation after interferon β (IFNβ) or IL-1β treatment of THP-1 cells. N = 4 independent experiments. d. % of infection in THP-1 cells after addition of IFNβ or IL-1β at different time points. Dotted line indicates the % of infection of untreated cells. N = 3 independent experiments e. Infection levels in MDM depleted of TRIM5 with equal genome copies of VSV-G pseudotyped HIV-1(M), HIV-2 or HIV-1(O) –GFP measured 48 h post-infection. N = 2 donors. Data shows mean + SD. Source data

Extended Data Fig. 5

a. Replication of HIV-1(M) NL4.3 (Bal Env) bearing HIV-2 ROD10 or HIV-1(O) MVP5180 Capsid in permissive GHOST cells (flow cytometry for induced GFP expression) representative of 2 independent experiments. b. Binding of fluorescently labeled nucleotides to HIV-1(M) or HIV-1(O) recombinant CA hexamers in the presence or absence of DTT to reduce monomer cross links. c. Titres of HIV-1(M), HIV-2 or HIV-1(O) -GFP made by mixing WT and CA R18G bearing packing constructs (left axis, white circles) and DNA synthesis measured at 6 hours post infection (right axis, black circles). d. Amino acids in BHP hinge region influencing BHP position with overlay of HIV-1(O) (PDB ID:7T12) and open HIV-1(M) (PDB ID:5HGL) hexamers. e. Capsid survival curves for CA mutant Q50Y generated from pooled data from two (no IP6) or three (100 μM IP6) independent experiments showing IP-mediated capsid stabilisation. f. Single round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) (R9) CA Q50Y –GFP measured 48 h post-infection by flow. Mean + /− SD, n = 3 independent experiments and 3 donors for MDM. Source data

Extended Data Fig. 6

a. Exterior view of the CA Q50Y/R120 hexamer (PDB ID: 8D3B) showing that the beta-hairpin (wheat) adopts an open conformation. b. Close-up view of the hinge region showing the H12-D51 salt-bridge necessary for the open conformation. The structure is indistinguishable from the Q50Y mutant in this region.

Extended Data Fig. 7

a, b. Secreted CXCL10 (A) IL8 (B) (ELISA) 48hpi and GAPDH-normalised mRNA levels in MDM (fold induction over uninfected 24hpi). c. % Infection of HIV-1(M) CA Q50Y 120 R in non-targeting CRISPR (NT-KO) or MAVS CRISPR knock out (MAVS-KO) cells. d. GAPDH-normalised mRNA levels, (fold induction over uninfected), in non-targeting CRISPR control (NT-KO) or MAVS KO THP-1 cells 24hpi. e. IRF-luciferase reporter 48 h after infection by equal genome copies of VSV-G-pseudotyped HIV-1(M) WT or Q50Y 120R mutant in ThP1 cells Ctrl or overexpressing TREX. f. Infection measured at 48 hours corresponding to (e) by flow. Mean + /− SD, n = 3 independent experiments or donors. g. VSV-G pseudotype titration curves in U87 cells expressing non-targeting control shRNA (shCtrl) or TRIM5 targeting shRNA (shTRIM5). Mean + /− SD n = 2 independent experiments. a, b two-tailed unpaired t-test vs untreated MDM. *p < 0.05, **p < 0.01. e, f paired t-test vs THP-1 Ctrl vector. ***p < 0.001. Source data