A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection
- Molecular and Cellular Biology Graduate Program, University of Washington, United States
- Division of Immunology and Pathogenesis, University of California, Berkeley, United States
- Divisions of Human Biology and Basic Sciences, Fred Hutchinson Cancer Center, United States
- Department of Microbiology, University of Washington, United States
Figures
Figure 1
The F59-F60 motif allows human CARD8 to detect protease activity from multiple HIV strs.
(A) Phylogenetic alignment of primate CARD8 protein sequences. The HIV protease (HIVPR) cleavage site is indicated by a purple triangle between F59 (P1) and F60 (P1’). Numbering is based on human CARD8. (B) Depiction of the mCherry-CARD8 used in cleavage assays in (C and D). The predicted molecular weights (kDa) for full-length, FIIND-processed, or HIVPR cleavage products are indicated. FIIND, function-to-find domain; CARD, caspase activation and recruitment domain. (C) HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged wildtype (WT) CARD8 and indicated HIV proviral constructs, HIV-1LAI, HIV-1Q23, or HIV-2ROD, in the presence (‘+’) or absence (‘–’) of 10 µM lopinavir, an HIVPR inhibitor. Top: Immunoblotting for anti-mCherry to detect the mCherry-CARD8 fusion protein. The full-length and FIIND-processed bands are indicated as well as the HIVPR cleavage product. The band at ~45 kDa is the result of cleavage by the 20S proteasome (Hsiao et al., 2022). Middle: Immunoblotting with an anti-p24gag antibody showing Gag cleavage products p41gag and p24gag, and/or full-length Gag, p55gag. Bottom: Immunoblotting with anti-vinculin as a loading control. (D) HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged WT, F60L, or F60S CARD8 and indicated HIV proviral constructs. Immunoblotting and labeling of the blots as in (C).
Figure 2 with 1 supplement
Natural variation in CARD8 alters sensing of SIVcpzPR activity.
(A) HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged human or chimpanzee (cpz) CARD8 and indicated provirus constructs. Immunoblotting was carried out for CARD8 cleavage, HIV/SIV protease (S/HIVPR) activity, and vinculin (loading control) as indicated. The S/HIVPR cleavage product is indicated by a purple triangle. FIIND, function-to-find domain. (B) HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged wildtype (WT), F60L, or F60S CARD8 and indicated proviral constructs. Immunoblotting was performed as in (A).
Figure 2—figure supplement 1
SIVmac cleaves wildtype (WT) human CARD8.
HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged WT or F60A human CARD8 and indicated provirus constructs. Immunoblotting was carried out for CARD8 cleavage, HIV/SIV protease (S/HIVPR) activity, and vinculin (loading control) as indicated. The anti-p24gag antibody (for S/HIVPR activity) poorly cross-reacts with SIVmac239. The S/HIVPR cleavage product is indicated by a purple triangle. We note that SIVmac239PR still partially cleaves CARD8 F60A. FIIND, function-to-find domain.
Figure 3 with 1 supplement
HIV-1 infection activates the CARD8 inflammasome in THP-1 cells.
(A) THP-1 cells were mock infected or infected with HIV-1LAI or HIV-1LAI-VSVG, yielding 8% and 53% p24gag+ cells after 24 hr, respectively. Immunoblotting using cytoplasmic lysates was carried out for HIV protease (HIVPR) activity, and vinculin (loading control) as indicated 24 hr post-infection. (B) THP-1 cells were either left unprimed or primed with different Toll-like receptor (TLR) agonists 4–6 hr before treatment with 10 µM VbP or infection with HIV-1LAI-VSVG at a multiplicity of infection (MOI) such that 30–50% were p24gag+ after 24 hr. Inflammasome responses were measured 24 hr following VbP treatment or HIV-1 infection. Left: Cell death is reported as the percent of propidium iodide (PI) positive cells. Right: Interleukin (IL)-1β levels were measured using the IL-1R reporter assay. (C) Wildtype (WT), CARD8 knockout (KO), or caspase-1 (CASP1) KO THP-1 lines were primed with Pam3CSK4 then challenged with either HIV-1LAI or HIV-1LAI-VSVG at an MOI such that 30–50% of WT cells were p24gag+ after 24 hr. Subsequent inflammasome activation was assayed 24 hr post-infection. Left and middle: Cell death and IL-1β levels were measured as in (A). Right: Active CASP1 was measured with CASP1-specific FLICA dye. (D) WT or CARD8 KO THP-1s overexpressing CCR5 were primed and treated with 10 µM VbP or infected with HIV-1BG505 for 24 hr such that ~30% of cells were p24gag+ then probed for inflammasome activation via IL-1β secretion. HIV-1BG505 is a CCR5 tropic strain in clade A. The dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=3 biological replicates). p-Values were determined by two-way ANOVA with Dunnett’s (B–C) or Sidak’s (D) test using GraphPad Prism 9. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.
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Figure 3—source data 1
Tables of source data for propidium iodide uptake, FLICA, and IL-1β secretion.
- https://cdn.elifesciences.org/articles/84108/elife-84108-fig3-data1-v2.zip
Figure 3—figure supplement 1
Functional validation of CARD8 knockout (KO) THP-1 cells.
(A) Immunoblot of wildtype (WT) or CARD8 KO THP-1 lines was carried out for CARD8 expression using endogenous antibody against CARD8 C-terminal domain (CTD) and loading control (vinculin). FIIND, function-to-find domain. (B) WT or CARD8 KO THP-1 cells were primed with Pam3CSK4 for 4–6 hr then treated with 10 µM VbP for 24 hr or 5 µg/mL nigericin for 3 hr before probing for interleukin (IL)-1β secretion (left) or propidium iodide (PI) dye uptake (right). The dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=3 biological replicates). p-Values were determined by two-way ANOVA Sidak’s test using GraphPad Prism 9. ns = not significant, *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.
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Figure 3—figure supplement 1—source data 1
Tables of source data for propidium iodide uptake and IL-1β secretion.
- https://cdn.elifesciences.org/articles/84108/elife-84108-fig3-figsupp1-data1-v2.zip
Figure 4
Incoming and outgoing HIVPR ar responsible for CARD8 inflammasome activation.
(A) Wildtype (WT), CARD8 knockout (KO), or caspase-1 (CASP1) KO THP-1 cells were primed overnight with Pam3CSK4 and then infected with HIV-1LAI-VSVG. Supernatant was collected at 0, 2, 4, 6, 8, and 24 hr post-infection to measure interleukin (IL)-1β secretion. Cells were infected at viral concentration such that ~70% of cells were p24gag-positive after 24 hr. (B) WT THP-1 cells primed with Pam3CSK4 were challenged with WT or mutant HIV-1LAI-VSVG or WT virus preincubated in 5 µM lopinavir (LPV) for 30 min prior to infection. HIV-1LAI-VSVG ΔVpr has a frameshift mutation in Vpr. Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=3 biological replicates). p-Values were determined by two-way ANOVA with Dunnett’s test using GraphPad Prism 9. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 4—source data 1
Tables of source data for IL-1β secretion.
- https://cdn.elifesciences.org/articles/84108/elife-84108-fig4-data1-v2.zip
Figure 5 with 1 supplement
HIV-1 inflammasome activation is dependent on a human-specific motif in CARD8.
(A) CARD8 knockout (KO) THP-1 lines complemented with different doxycycline (dox)-inducible CARD8 variants (pCARD8) were left uninduced or induced for 18 hr. Immunoblot of wildtype (WT) or complemented CARD8 KO THP-1 lines treated with (‘+’) or without (‘–’) dox was carried out for CARD8 expression using endogenous antibody against CARD8 C-terminal domain (CTD) and loading control (vinculin). FIIND, function-to-find domain. (B–C) Complemented CARD8 KO lines were left uninduced or dox-induced as described in (A) and then primed for 4–6 hr with Pam3CSK4 and treated with either 10 µM VbP or HIV-1LAI-VSVG then assessed for (B) interleukin (IL)-1β secretion and (C) cell death, respectively. (D) CARD8 KO THP-1 lines complemented with different CARD8 variants were left uninduced or induced for 18 hr. Immunoblot of wildtype (WT) or complemented CARD8 KO THP-1 lines treated with (‘+’) or without (‘–’) dox as described in (A). (E–F) Complemented CARD8 KO lines were induced and primed as described in (B) then treated with either 10 µM VbP or HIV-1LAI then assessed for (E) IL-1β secretion and (F) cell death, respectively. All HIV infections were done at a multiplicity of infection (MOI) such that 30–50% of cells were p24gag+ after 24 hr. Dotted line indicates limit of detection (LoD). Datasets represent mean ± SD (n=3 biological replicates). p-Values were determined by two-way ANOVA with Tukey’s test using GraphPad Prism 9. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 5—source data 1
Tables of source data for propidium iodide uptake and IL-1β secretion.
- https://cdn.elifesciences.org/articles/84108/elife-84108-fig5-data1-v2.zip
Figure 5—figure supplement 1
Human FF motif in chimpanzee CARD8 rescues sensing of HIV/SIVPR.
HEK293T cells were transfected with a construct encoding N-terminally mCherry-tagged human, chimpanzee (cpz), or cpz with the human FF motif (L60F) CARD8 and indicated provirus constructs. Immunoblotting was carried out for CARD8 cleavage, HIV/SIV protease (S/HIVPR) activity, and vinculin (loading control) as indicated. The S/HIVPR cleavage product is indicated by a purple triangle. FIIND, function-to-find domain.
Additional files
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Supplementary file 1
Primers, reagents, and primate CARD8 sequence info.
(a) List of primers, gBlocks, and sgRNA sequences. (b) List of antibodies/reagents. (c) Primate CARD8 gene IDs.
- https://cdn.elifesciences.org/articles/84108/elife-84108-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84108/elife-84108-mdarchecklist1-v2.pdf
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Source data 1
- https://cdn.elifesciences.org/articles/84108/elife-84108-data1-v2.zip
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Source data 2
- https://cdn.elifesciences.org/articles/84108/elife-84108-data2-v2.pdf
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Source data 3
Uncropped western blots pdf from Figure 2—figure supplement 1, Figure 3—figure supplement 1, Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/84108/elife-84108-data3-v2.pdf
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A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection
eLife 12:e84108.
https://doi.org/10.7554/eLife.84108
