Abstract
At steady state, the NOD-like receptor (NLR)-containing pyrin domain (PYD) (NLRP)1 inflammasome is maintained in an auto-inhibitory complex by dipeptidyl peptidases 8 and 9 (DPP8 and DPP9) and is activated by pathogen-encoded proteases after infection. Here, we showed that the open reading frame (ORF)45 protein of the Kaposi’s sarcoma-associated herpesvirus activated the human NLRP1 (hNLRP1) inflammasome in a non-protease-dependent manner, and we additionally showed that the Linker1 region of hNLRP1, situated between the PYD and NACHT domains, was required for the auto-inhibition and non-protease-dependent activation of hNLRP1. At steady state, the interaction between Linker1 and the UPA subdomain silenced the activation of hNLRP1 in auto-inhibitory complexes either containing DPP9 or not in a manner independent of DPP9. ORF45 binding to Linker1 displaced UPA from the Linker1–UPA complex and induced the release of the C-terminal domain of hNLRP1 for inflammasome assembly. The ORF45-dependent activation of the NLRP1 inflammasome was conserved in primates but was not observed for murine NLRP1b inflammasomes.
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All data are available in the article and supplementary files or from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank J. U. Jung, K. Lan, X. Liang, J. Zhang, D.A. Leib, D. Pan, B. Du, J. Han, B. Fu, G. Long, G. Zou, N. Dong and F. Shao for providing plasmid constructs and different virus strains. We thank F. Shao (National Institute of Biological Sciences, China) for valuable suggestions. The Core Facility of Basic Medical Sciences at the Shanghai Jiao Tong University School of Medicine provided the cDNA plasmids for cloning. This work was supported by grants from the National Key Research and Development Project of China (2018YFA0900802, Q.L.), the Shanghai Science and Technology Commission (22ZR1454500, Q.L.), the Shanghai Frontiers Science Center of Cellular Homeostasis and Human Disease (Q.L.), the Shanghai Municipal Science and Technology Major Project (ZD2021CY001, Q.L.), the Innovative Research Team of High-level Local Universities in Shanghai (Q.L. and Z.L.) and the Shanghai Municipal Health Commission (201940179, Z.L.).
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Q.L. conceived the research, designed the study and wrote the manuscript. X.Y., J.Z., C.L., Y.Q., W.W. and Z.L. performed experiments and analyzed data. F.Z. generated the iSLK-BAC16 ORF45-expressing stop cell line. M.Z.X.X. helped with the visual representation of data and edited the manuscript. All authors commented on the manuscript.
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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 KSHV ORF45 triggers inflammasome activation.
a,b ASC spike microscopy images (a) and its quantification (b) in GFP-ASC HeLaWT or HeLaIFI16 KO cells infected by KSHV. Scale bar, 50 μm. n = 4. c IFI16 knockout in HeLa cell by CRISPR-Cas9. d ASC spike microscopy images in GFP-ASC HeLa cells at 48 h after infection with lentiviruses carrying individual KSHV-encoded protein. Scale bar, 50 μm. e,f IL-1β production (e) and LDH release (f) in THP-1 cells infected by KSHVWT, KSHVUV, or KSHVΔORF45 for 24 h (n = 3). g LDH release of HEK293T cells expressing Tet-vector or Tet-ORF45 treated with 1 μg/ml Dox for 48 h (n = 3). h Fluorescence microscopy images in THP-1 cells with TAT-GFP treatment for 2 h. Scale bar, 50 μm. All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, b,g two-tailed paired Student’s t test. e,f one-way ANOVA.
Extended Data Fig. 2 hNLRP1 is required for ORF45-mediated inflammasome activation.
a Endogenous expressions of hNLRP1, NLRP3, NLRC4, IFI16 and Pyrin in THP-1, BJAB, HEK293T and HeLa cells by immunoblot with indicated antibodies. b,c ASC spike microscopy images (b) and its quantification (c) in GFP-ASC HeLaWT or HeLaNLRP1 KO cells infected by KSHV for 48 h. Scale bar, 50 μm. n = 4. d,e ASC spike microscopy images (d) and its quantification (e) in GFP-ASC HeLa cells infected by lentiviruses carrying ORF45, 3CPro, and vector control for 48 h. Scale bar, 50 μm. n = 3. f-h LDH release (f,g) and IL-1β production (h) in THP-1WT or THP-1NLRP1 KO cells treated with TAT-GFP, TAT-ORF45, TAT-3Cpro (10 μg/ml), or poly I:C (500 ng/ml) for 6 h (n = 3). All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, c,g,h two-tailed paired Student’s t test. e,f one-way ANOVA.
Extended Data Fig. 3 KSHV ORF45 interacts with hNLRP1.
a-c Two-hybrid interaction assay in yeast transformed with expression vectors for indicated combinations of KSHV-encoded proteins and inflammasome components. d Nuclear and cytoplasm fractions were generated from HeLa-hNLRP1-Flag stable cells infected with lentiviruses carrying ORF45 or vector for 48 h, followed by immunoprecipitation (IP) and immunoblot (IB) with indicated antibodies. e,f Interaction assay between ORF45 and indicated hNLRP1 mutants in yeast by two-hybrid interaction assay (e) or in HEK293T cells transfected with indicated expression vectors by co-IP (f). g Two-hybrid interaction assay in yeast transformed with expression vectors for indicated combinations of ORF45 and Linker1 mutants. h,i ASC spike microscopy images (h) and its quantification (i) in GFP-ASC HeLa cells infected by lentiviruses carrying vector, ORF45, or ORF45 mutants for 48 h. Scale bar, 50 μm. n = 6. j Schematic diagram of ORF45-hNLRP1 specific interaction. The left panel shows a phylogenetic tree of the analyzed species with hominoids in black text, Old World monkeys in green text, and New World monkeys in blue text. The right panel shows the alignment of a portion of the Linker1 region. Amino acid labelled in red color differ from hNLRP1. All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, i one-way ANOVA.
Extended Data Fig. 4 hNLRP1 forms DPP9-dependent and DPP9-independent auto-inhibitory complexes.
a-c HEK293T cells were co-transfected with expression vectors for indicated combinations, followed by IP and IB with indicated antibodies. VbP: 10 μM for 6 h for (b). d Two-hybrid interaction assay in yeast transformed with expression vectors for various combinations of hNLRP1 fragments. e-i HEK293T cells were co-transfected with expression vectors for indicated combinations, followed by IP and IB with indicated antibodies. VbP: 10 μM for 6 h for (h). j p17 generation in ASC-caspase-1-pro-IL-1β HEK293T cells transfected with indicated expression vectors for 36 h. k-m ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h. n = 5. n p17 generation in ASC-caspase-1-pro-IL-1β HEK293TWT or HEK293TDPP8/9 DKO cells transfected with indicated expression vectors for 36 h. o ASC spike quantification in GFP-ASC HEK293TWT or HEK293TDPP8/9 DKO cells transfected with indicated expression vectors for 24 h. n = 3. p ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h. n = 5. All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, k-m,o,p one-way ANOVA.
Extended Data Fig. 5 KSHV ORF45 activates hNLRP1 inflammasome via Linker1 region.
a Inhibition of hNLRP1NT-hNLRP1CT association by ORF45Δ300-332 via co-IP assay. b ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h. n = 5. c ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h in the presence or in the absence of VbP (10 μM for 6 h). n = 3. d p17 generation in ASC-caspase-1-pro-IL-1β HEK293T cells transfected with indicated expression vectors for 48 h. e ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h. n = 5. f p17 generation in ASC-caspase-1-pro-IL-1β HEK293TWT or HEK293TDPP8/9 DKO cells transfected with indicated expression vectors for 36 h. g ASC spike quantification in RFP-ASC HEK293TWT or HEK293TDPP8/9 DKO cells transfected with indicated expression vectors for 24 h. n = 5. h ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h in the presence or in the absence of MG132 treatment (10 μM for 6 h). n = 3. i,j LDH release in THP-1 cells treated with MG132 (10 μM), Bortezomib (0.5 μM), or MLN4924 (1 μM) for 12 h, followed by TAT-ORF45 or VbP treatment for 6 h. n = 3. k ASC spike quantification in GFP-ASC HEK293T cells transfected with indicated expression vectors for 24 h in the presence or in the absence of Bortezomib (0.5 μM) or MG132 treatment (10 μM) for 6 h. n = 6. All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, b,c,e,g,h,k one-way ANOVA.
Extended Data Fig. 6 KSHV ORF45 activates primate NLRP1 inflammasome.
a Expression of indicated genes in GFP-ASC HEK293T cells in Fig. 6a. b,c p17 generation in ASC-caspase-1-pro-IL-1β HEK293T cells transfected with indicated expression vectors for 36 h. d,e p17 generation in ASC-caspase-1-pro-IL-1β HEK293T cells transfected with the expression vectors for indicated combination of ORF45, NLRP1 fragments from rhesus (d), or NLRP1 fragments from saimiri (e) for 36 h in the presence of in the absence of VbP (10 μM) or MG132 (10 μM) for 6 h. All data represent three independent experiments.
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Yang, X., Zhou, J., Liu, C. et al. KSHV-encoded ORF45 activates human NLRP1 inflammasome. Nat Immunol 23, 916–926 (2022). https://doi.org/10.1038/s41590-022-01199-x
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DOI: https://doi.org/10.1038/s41590-022-01199-x
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