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[Preprint]. 2024 May 10:2024.04.22.590513.
doi: 10.1101/2024.04.22.590513.

Cell-free assays reveal the HIV-1 capsid protects reverse transcripts from cGAS

Tiana M Scott  1 , Lydia M Arnold  1 , Jordan A Powers  1 , Delaney A McCann  1 , Devin E Christensen  2 , Miguel J Pereira  2 , Wen Zhou  3 , Rachel M Torrez  2 , Janet H Iwasa  2 , Philip J Kranzusch  4   5 , Wesley I Sundquist  2 , Jarrod S Johnson  1
Affiliations

Cell-free assays reveal the HIV-1 capsid protects reverse transcripts from cGAS

Tiana M Scott et al. bioRxiv. .

Abstract

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect their genomes from cGAS even after completion of reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, capsid inhibitors also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

Keywords: DNA sensing; HIV; IRF3; STING; cGAS; capsid inhibition; in vitro reverse transcription; innate immunity; interferon; lenacapavir.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. The HIV-1 capsid protects RT products from cGAS in vitro.
(A) Steps in the endogenous reverse transcription (ERT) and cell-free sensing assays. Capsids that remain intact after ERT may protect viral DNA from detection and capsids that lose integrity may permit cGAS activation and cGAMP production. (B) qPCR measurements of plasmid DNA control containing the viral genome sequence compared to HIV-1 ERT reactions in 2-fold dilution series, incubated for 16 h under identical conditions. Primer sets detect sequences for early (minus strand strong stop), intermediate (first strand transfer), and late RT (second strand transfer), which are present in both the plasmid and viral genome. (C) Cell-free sensing assay for samples shown in (B) incubated with recombinant cGAS. cGAMP levels were determined by ELISA (applies to all cell-free sensing assays). (D) Cell-free sensing assay of heat treated ERT samples. Samples were held at the indicated temperatures (20 s) and then subject to cell-free sensing assays performed under standard conditions. (E) Cell-free sensing assay of ERT samples incubated with cGAS under a range of IP6 concentrations. (F) qPCR measurements of DNA copy numbers from ERT reactions with wt HIV-1 virions compared to virions with stabilizing (E45A) or destabilizing (Q63/67A) mutations in CA. (G) Cell-free sensing assay of wt, E45A, or Q63/67A ERT samples after normalizing for DNA input and incubating with cGAS at the indicated IP6 concentrations. Statistics were calculated using a one-way ANOVA with Tukey’s multiple comparisons test: p<0.05: *, p<0.01: **, p<0.001: ***, p<0.0001: ****. Graphs depict mean ± SD from three samples from a representative experiment, selected from three independent experiments.
Figure 2.
Figure 2.. Lenacapavir triggers cGAS sensing of HIV-1 reverse transcripts.
(A) DNA product levels from an HIV-1 ERT time course compared to a plasmid control, as measured by qPCR (top panel), and cell-free sensing assays for the same samples incubated with recombinant cGAS ±LEN (100 nM) (bottom panel). (B) DNA products (top panel) and cell-free cGAS activity (bottom panel) for ERT reactions performed in the presence of increasing concentrations of the RT inhibitor efavirenz (EFV), or without dNTPs, or using a virus containing an inactivating mutation in RT (D185A). Statistics were calculated using a one-way ANOVA with Tukey’s multiple comparisons test: p<0.001: ***, p<0.0001: ****. Graphs depict mean ± SD from three samples from a representative experiment, selected from three independent experiments.
Figure 3.
Figure 3.. CA mutations confer resistance to LEN-triggered cGAS activation.
(A) qPCR measurements of DNA copy number from ERT reactions with wt HIV-1 virions compared to virions bearing LEN resistance-associated mutations in CA (M66I and Q67H/N74D), performed with or without dNTPs. (B) Cell-free sensing assay of wt, M66I, or Q67H/N74D ERT samples that were incubated with increasing amounts of LEN (0, 1, 3.2, 10, 32, 100 nM), or LEN (100 nM) −dNTPs. (C) qPCR measurements of DNA copy number for ERT reactions held at 37°C for the indicated times. (D) Cell-free sensing assay of ERT samples from (C) after normalizing for late RT DNA copy number and reacting with recombinant cGAS (±LEN at 100 nM). Statistics were calculated using a one-way ANOVA with Tukey’s multiple comparisons test: p<0.05: *, p<0.0001: ****. Graphs depict mean ± SD from three measurements from a representative experiment, selected from 3 independent experiments (A–C) or from 7 independent samples measured on different days (D).
Figure 4.
Figure 4.. Capsid inhibitors potentiate sensing of HIV-1 through the cGAS-STING pathway in cells.
(A) qPCR measurements of DNA copy number from ERT reactions with the indicated concentrations of LEN (top panel) and cell-free sensing assays (bottom panel) performed with the same samples and the same concentrations of LEN. (B) Flow cytometry of THP-1 monocytic cells infected with HIV-1-GFP for 48 h (±LEN at 10 nM), showing expression of GFP (viral infectivity) and SIGLEC1 (IFN response). (C) Heat maps of GFP, ISG15, and SIGLEC1 expression as determined by flow cytometry of THP-1 cells infected with HIV-1-GFP for 48 h. Columns show a range of LEN concentrations and rows show increasing virus doses (12.5, 25, and 50 nM p24). n = 3. (D) Immunoblots of THP-1 lysates after lentiCRISPR editing, depicting knockout of cGAS, STING, or IRF3 relative to a non-targeting vector control (LCV2). (E–F) Flow cytometry of lentiCRISPR-modified THP-1 cells infected with HIV-1-GFP (25 nM p24) for 48 h, showing the percent of cells positive for GFP (E) or SIGLEC1 (F) expression for the indicated conditions (±LEN at 10 nM). (G–H) Flow cytometry of MDDCs derived from 4 separate donors that were infected with HIV-1-GFP for 48 h with increasing concentrations of GS-CA1. Vpx was included to overcome SAMHD1-mediated restriction. Panels show infectivity (GFP+, G) and IFN induction (ISG15+, H). (I–J) Flow cytometry of MDMs from 4 donors that were infected with HIV-1-GFP in the same fashion as in (G–H) without Vpx. Graphs depict the percentage of GFP (G,I) or ISG15 (H,J) positive cells. For (A,F), statistics were calculated using a one-way ANOVA with Tukey’s multiple comparisons test: p<0.05: *, p<0.001: ***, p<0.0001: ****. Graphs depict mean ± SD from three samples from a representative experiment, selected from 2–3 independent experiments, unless otherwise noted.
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