CARD8 inflammasome activation during HIV-1 cell-to-cell transmission

Jessie Kulsuptrakul
Michael Emerman author has email address
Patrick S Mitchell author has email address

Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, USA
Divisions of Human Biology and Basic Sciences, Fred Hutchinson Cancer Center, Seattle, USA
Department of Microbiology, University of Washington, Seattle, USA
Howard Hughes Medical Institute, University of Washington, Seattle, USA

https://doi.org/10.7554/eLife.102676.1

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Figures and data

HIV-1 cell-to-cell infection induces inflammasome activation.
(A) Wildtype (WT) or CARD8 knockout (KO) THP-1 cells were infected with HIV-1_LAI at the same MOI in the presence or absence of DEAE-dextran (10µg/mL) then harvested after 24 hours and assayed for: left) percent infection via intracellular p24^gag, middle) inflammasome activation by IL-1β secretion via IL-1R reporter assay, and right) cell death via propidium iodide (PI) dye uptake using flow cytometry. (B) SUPT1 or THP-1 cells were primed with Pam3CSK4 (500ng/mL) overnight then treated with 5μM ValboroPro (VbP) for 24 hours then assessed for IL-1β secretion as in (A). (C) Schematic illustrating experimental setup for SUPT1:THP-1 cell coculture. (D) THP-1 cells were primed overnight with Pam3CSK4 prior to coculture. SUPT1 cell were either mock infected or infected with HIV-1_LAI then cocultured with primed WT THP-1 cells 20 hours post infection. Mock- or HIV-1_LAI-infected SUPT1 cells were either mixed with the THP-1 cells or put in a transwell with a virus-permeable membrane in the presence or absence of DEAE-dextran. DEAE-dextran was added to the mock but not the mixed condition. Supernatant from the cocultures were harvested 3 days after starting the coculture and assayed for IL-1β secretion as in A) and B). Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (A-B: n=2; D n=4 biological replicates). A-B: Two-way ANOVA with Tukey’s test D: One-way ANOVA with Tukey’s test using GraphPad Prism 10. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

HIV-1 cell-to-cell infection induces inflammasome activation.
(A) SUPT1 or THP-1 cells were primed with Pam3CSK4 (500ng/mL) overnight then treated with 5μM ValboroPro (VbP) for 24 hours then assessed for cell death via propidium iodide (PI) uptake. (B) SUPT1 cells were cocultured with THP-1 cells as in Figure 1D and supernatant in the cell-to-cell condition and in the supernatant outside of the transwell was sampled and measured for cell-free infectious HIV virions via reverse transcriptase (RT) assay. Datasets represent mean ± SD (A: n=2; B: n=4 biological replicates). B: Two-way ANOVA with Tukey’s test using GraphPad Prism 10. ns = not significant

HIV-1 cell-to-cell transmission induces CARD8-dependent activation largely independent of NLRP3.
(A) SUPT1 cells were either mock-infected or infected with HIV-1_LAI for 18-20 hours prior to coculture with wildtype (WT) or CARD8 knockout (KO) THP-1 cells. The coculture was harvested 72 hours later to probe for IL-1β secretion in the coculture supernatant via IL-1R reporter assay. THP-1 cells were primed with Pam3CSK4 (500ng/mL) for 16-24 hours prior to coculture. SUPT1 cells were infected with HIV-1_LAI such that 30% of the cells were positive for intracellular p24^gag after 18-20 hours. (B) SUPT1 cells were either mock or HIV-1_LAI-infected as in (A) for 18-20 hours then incubated in DMSO, lopinavir (LPV), MCC950, or VX765 at 0.01%, 5μM, 10μM, or 1μg/mL, respectively, for 15 minutes prior to coculturing with primed WT THP-1 cells. The coculture was assessed for subsequent inflammasome activation after 72 hours as in (A). Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=3 biological replicates). Two-way ANOVA with Dunnett’s test using GraphPad Prism 10. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

HIV-dependent inflammasome activation is largely NLRP3- independent.
(A) Wildtype THP-1 cells were pre-treated with either DMSO, lopinavir (LPV), MCC950, or VX765 at 0.01%, 5μM, 10μM, or 1μg/mL, respectively, for 15 minutes prior to 4-hour treatment with 5μg/mL nigericin. Subsequent inflammasome activation was assessed via (A-left) IL-1β secretion via IL-1R reporter assay and (A-right) cell death via propidium iodide (PI) dye uptake. (B) Wildtype THP-1 cells were pre-treated with indicated inhibitors as in (A) then infected with either HIV-1_LAI or increasing amounts of VSV-G pseudotyped HIV-1_LAI (HIV-1_LAI- _VSVG) in the presence of 10μg/mL DEAE-dextran. Subsequent inflammasome activation was assessed 24 hours post infection via IL-1β secretion and cell death as in (A). Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (A: n=3, B: n=2 biological replicates). One-way (A) or Two-way (B) ANOVA with Dunnett’s test using GraphPad Prism 10. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Cell-to-cell HIV infection induces CARD8-dependent activation in monocyte-derived macrophages (MDMs).
(A) MDMs were cocultured with SUPT1 cells expressing CCR5 (SUPT1-CCR5) that were mock-, HIV-1_LAI- or HIV-1_NL4.3-BaL-infected then assayed for inflammasome activation 48 hours post-coculture for IL-1β secretion. Fifteen minutes before starting the coculture, SUPT1-CCR5 cells infected with HIV-1_NL4.3-BaL were pre-treated with either DMSO, lopinavir (5μM), nevirapine (50μM), MCC950 (10μM ) or VX765 (1μg/mL), inhibiting HIV-1 protease (HIV^PR), HIV-1 reverse transcriptase (HIV^RT), NLRP3, or caspase 1 (CASP1), respectively. SUPT1-CCR5 cells were infected with HIV-1_LAI and HIV-1_NL4.3-BaL such that 2% and 7% of cells, respectively, were positive for intracellular p24^gag after 24 hours. (B) MDMs from 3 independent healthy donors were knocked out (KO) for either AAVS1 or CARD8 using a Synthego gene KO kit and were immunoblotted using an anti-CARD8 antibody or anti-vinculin. Full-length and FIIND-processed CARD8 intermediates are marked with a purple arrow. Table between CARD8 and vinculin blot shows Synthego gene KO% scores for each donor KO line. (C) AAVS1 and CARD8 KO MDM lines were primed with Pam3CSK4 (500ng/mL) overnight and then treated with ValboroPro (VbP) for 24 hours before assessing for IL-1β secretion. (D) AAVS1 and CARD8 KO MDM lines were cocultured with SUPT1-CCR5 cells mock-, or HIV-1_NL4.3-BaL-infected then assayed for inflammasome activation 48 hours post-coculture for IL-1β secretion. Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (A: n=3 independent donors, C-D: n=3 technical replicates per donor). (A) One-way or (D) two-way ANOVA with Tukey’s test (using GraphPad Prism 10. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Protease inhibitor resistance mutations and relative CARD8 cleavage.

Protease inhibitor resistant strains of HIV-1 differentially cleave and activate CARD8.
(A) HEK293T cells were transfected with a construct encoding CARD8 with an N-terminal mCherry tag (mCherry-CARD8) and indicated HIV-1 proviral constructs. Protease inhibitor-resistant (PI-R) clones of HIV-1 are a subset of a panel expressing prototypical multidrug resistant HIV-1 protease (HIV^PR) in an NL4.3 backbone (Table S1). Top: Immunoblotting using anti-mCherry antibody to detect mCherry-CARD8. The full-length (mc-CARD8) and FIIND-processed bands are indicated as well as the HIV^PR cut product. The band at ∼45 kDa is the result of cleavage by the 20S proteasome. % CARD8 cleavage was calculated by quantifying the HIV^PR cut band relative to the HIV-1_LAI control using BioRad Image Lab 6. Middle: Immunoblotting with an anti-p24^gag antibody showing HIV-1^gag cleavage products p41^gag and p24^gag, and/or full-length HIV-1^gag, p55^gag. %p24/p55 was calculated from the ratio of p24^gag versus p55^gag product by quantifying the volume of the p24^gag bands versus the p55^gag band relative to the HIV-1_LAI control using BioRad Image lab 6. Bottom: Immunoblotting with an anti-vinculin antibody to detect vinculin as a loading control. (B) HEK293T cells were transfected with human caspase 1 and human pro-IL-1β, and either carrier vector or indicated HIV-1 proviruses then probed for IL-1β secretion 24 hours post-transfection via IL-1R reporter assay. (C) HEK293T cells were transfected with indicated HIV-1 proviruses (300ng). 24 hours post-transfection either wildtype (WT) or CARD8 knockout (KO) THP-1s were overlayed on the transfected HEK293T cells in a 1:1 ratio. THP-1s were primed with Pam3CSK4 overnight prior to coculture. Supernatants were harvested 24 hours post coculture to assay for IL-1β secretion as in (B). Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=4 biological replicates). p-Values were determined by two-way ANOVA with Dunnett’s test using GraphPad Prism 10. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

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