Abstract
Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1,2,3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.
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Data availability
All data are available in the main paper, supplementary information and via figshare https://doi.org/10.6084/m9.figshare.21586098. All reagents are available from the authors under a material transfer agreement with University of Texas Southwestern Medical Center. Source data are provided with this paper.
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Acknowledgements
The authors thank Z. G. Wang for providing the pLVX-TRE3G vector; J. Rizo-Rey, Z. G. Wang and K. Yang for helpful discussions and technical assistance; and members of the Reese laboratory for technical assistance. T.A.R. is supported by grants from the NIH (R01AI130020-01A1, U19AI142784), CPRIT (RP200118), and the Pew Scholars Program. D.C.H. is funded by a 1R35GM142689-01 and a Recruitment of First-Time, Tenure-Track Faculty from Cancer Prevention & Research Institute of Texas Award (RR 170047).
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G.W. and T.A.R. conceived the study. G.W. and D.Z. performed experiments and analysed data. D.C.H. performed sequence and phylogenetic analysis. R.C.O. provided reagents and expertise. G.W. and T.A.R. wrote the paper. All authors read and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Noroviruses infection triggers programmed cell death.
a, Viability of BMDMs and BV2 infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. b, LDH released from BMDMs and BV2 infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. c, Representative flow cytometry pseudocolor dot plots of propidium iodide (PI) and annexin V-stained BMDMs infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. Cells were first gated off forward and side scatter. d, Bright-field images of BMDMs and BV2 infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. Scale bar, 100 μm. e, Representative IncuCyte images of SytoxGreen-stained BMDMs and BV2 infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 24 h. Scale bar, 400 μm. Data in (a and b) are pooled from three independent experiments with three technical replicates in each experiment. Data are presented as mean of biological replicates ± s.d. Data in (c,d and e) are representative of three independent experiments.
Extended Data Fig. 2 Blockade of apoptosis, pyroptosis, or necroptosis does not rescue MNV-induced cell death in in BMDMs and BV2 cells.
a, Representative bright-field images of BMDMs infected with MNoVCR6 or MNoVCW3 at a MOI of 5 with or without 10 μM zVAD and/or 10 μM necrostatin-1 (Nec-1) for 12 h. Scale bar, 100μm. (b to c) Cytotoxicity of BMDMs infected with MNoVCR6 (b) or MNoVCW3 (c) at a MOI of 5 with or without 10 μM zVAD and/or 10 μM Nec-1 for 24 h. d, Representative flow cytometry pseudocolor dot plots of propidium iodide (PI) and annexin V-stained BMDMs are shown. BMDMs infected with MNoVCW3 and MNoVCR6 at a MOI of 5 with or without 10 μM zVAD and/or 10 μM Nec-1 for 12 h. Cells were first gated off forward and side scatter. e – h, LDH released from BMDMs (e and f) and BV2 (g and h) infected with MNoVCW3 and MNoVCR6 (MOI=5) with or without 10 μM zVAD, 10 μM Nec-1, either individually or in combination for 24h. i, Cytotoxicity of BV2 cells infected with MNoVCR6 at a MOI of 1 with or without zVAD for 12 h. BV2 cells treated with 0.5 μM Staurosporine (STS) with or without 20 μM zVAD for 6 h as a positive control for apoptosis. j, BV2 cells were primed with 200 ng/mL LPS for 4 h, followed by 10 μM nigericin (Niger) to activate pyroptotic cell death, or they were infected with MNoVCR6, MOI = 1, 12 h. zVAD (20 μM) was added 30 min before nigericin treatment. After 12h treatment or infection, cell viability measured by ATP levels using CellTiter-Glo kit. k, Cytotoxicity of BV2 cells infected with MNoVCR6 at a MOI of 1 with or without 10 μM Nec-1 for 12 h. BV2 cells treated with 20 μM LPS plus 20 μM zVAD to induce necroptosis and treated with or without 30 μM Nec-1 as a control. After 12h treatment or infection, cell viability measured by ATP levels using CellTiter-Glo kit. Data in (a and d) are representative of three independent experiments. Data in (b,c,e,f,g,h,i,j and k) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d.
Extended Data Fig. 3 Pyroptosis, necroptosis and apoptosis are not required for MNoV-induced cell death.
a, WT and Caspase1/11−/− BMDMs cells were treated with vehicle, 10 μM Nec-1 and infected with MNoVCW3 and MNoVCR6 at a MOI of 1 for 24 h. Cell viability measured by ATP levels. b, GSDMD was deleted from BV2 cells by CRISPR genome editing. GSDMD expression levels in WT and four different clones of knockout BV2 cells. Lysates of indicated cells were blotted with anti-GSDMD antibody and anti-actin antibody. c, Comparison of LDH release in WT and Gsdmd−/− cells infected with MNoVCW3 and MNoVCR6 (MOI = 1) for 24 h. d WT and Gsdmd−/− cells infected with MNoVCW3 and MNoVCR6 (MOI = 1) for 24 h. Cell viability as measured by ATP levels. e, WT BMDMs cells infected with MNoVCW3 and MNoVCR6 at a MOI of 1 or treated with 10 μM zVAD plus 100 ng/ml LPS. Lysates of indicated cells were blotted with anti-MLKL antibody, anti-pMLKL antibody and anti-actin antibody. f, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI = 1) infected WT and Ripk3−/− BMDMs with or without 10 μM zVAD for 24 h. g, MLKL was deleted from BV2 cells by CRISPR genome editing. Lysates of wildtype of a knockout clone of MLKL were blotted with anti-MLKL antibody and anti-actin antibody. h, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI=1) infected WT and Mlkl−/− cells with or without 10 μM zVAD for 24 h. i, Bax and Bak were deleted from BV2 cells by CRISPR genome editing and single cell clones were selected. Lysates of WT or knockout cells were blotted with anti-BAK, anti-BAX and anti-actin antibody. j, Gsdmd/Caspase-3 and Gsdmd/Caspase-9 double knockout BV2 cells were made by CRISPR genome editing and single cell clones were selected. Lysates of indicated cells were blotted with indicated antibodies. k, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI = 1) infected WT and Bax−/− / Bak−/− cells for 12 h. l, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI = 1) infected WT, Gsdmd−/− / Caspase-3−/− and Gsdmd−/− / Caspase-9−/− cells for 12 h. m, WT BMDMs cells infected with MNoVCW3 and MNoVCR6 at a MOI of 1 or treated with 10 μg/mL Tunicamycin for 4 h. Lysates of indicated cells were blotted with anti-CHOP antibody, anti-ATF4 antibody, anti-IRE1a antibody and anti-actin antibody. n, WT and Ire1a−/− BV2 cells infected with MNoVCW3 and MNoVCR6 (MOI = 1) for 24. Cell viability as measured by ATP levels. Data in (a,c,d,f,h,k,l and n) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d. Data in (b,e,g,i,j and m) are representative of two independent experiments.
Extended Data Fig. 4 Norovirus NTPase NS3 directly triggers cell death.
a, Schematic model of MNoV NS3. b, Representative bright-field images of HEK293T cells transfected with N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C of MNoVCR6 or MNoVCW3. Scale bar, 100 μm. c, LDH released from HEK293T cells transfected with Full-length Flag-NS3 of MNoVCR6 or MNoVCW3. d, LDH released from HEK293T cells transfected with Full-length, N- or C-terminal Flag-NS3 of MNoVCR6. e, LDH released from HEK293T cells transfected with Full-length, N- or C-terminal Flag-NS3 of MNoVCW3. f, The immunoblot shows expression of HEK293T cells transfected with Full-length, N- or C-terminal of Flag-NS3. g, Immunoblotting assays of norovirus infection-induced GSDMD and CASPASE-3 cleavage in BMDMs. BMDMs infected with MNoVCW3 and MNoVCR6 (MOI = 5) for 12 h. h, Immunoblotting assays of GSDMD and CASPASE-3 cleavage in BV2 cells stably expressing inducible, C-terminal Flag-tagged constructs of NS3-FL, NS3-N or NS3-C of MNoVCR6 in the absence or presence doxycycline for 12 h. Data in (b) are representative of three independent experiments. Data in (c,d and e) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d. Data in (f,g and h) are representative of two independent experiments.
Extended Data Fig. 5 MNoV and HNoV NS3 N-terminal domain directly triggers cell death in multiple cell types.
NS3-FL, NS3-N or NS3-C fused with C-terminal Flag tag were stably expressed in BV2 cells under a tetracycline-inducible promoter. Protein expression was induced with doxycycline treatment. a, Representative bright-field images of BV2 cells stably expressing inducible constructs in the absence or presence doxycycline for 12 h. Scale bar, 100 μm. b, Immunoblot of NS3 with anti-Flag antibody in the absence or presence doxycycline for 12 h. c, Cytotoxicity of BV2 cells expressing inducible constructs after doxycycline addition with or without 20 μM zVAD for 12 h. d, Representative Incucyte images of SytoxGreen-stained stably BV2 cells after doxycycline addition for 24 h. Scale bar, 200 μm. e, BV2 cells expressing inducible constructs of NS3-FL, NS3-N and NS3-C. The time-course Incucyte quantification of SytoxGreen-positive cells in the absence or presence doxycycline. f and g, N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C from MNoVCR6 were transfected into A20 B cells (f) and HeLa cells (g). Cell death was determined by ATP-based cell viability assay 24 h after transfection. h, LDH released from HEK293T cells transfected with N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C of HNoV MD145. i, Representative bright-field images of HEK293T cells transfected with N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C of HNoV MD145. Scale bar, 100 μm. j, The immunoblot shows expression of transfected FL-, N- or C-NS3 of MD145. Data in (a,b,d,e,i and j) are representative of three independent experiments. Data in (c,f,g and h) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d.
Extended Data Fig. 6 Subcellular localization prediction of NS3 and AlphaFold prediction of NS3 and mouse MLKL N-terminal domain.
DeepLoc prediction server revealed that full-length NS3 of (a) HNoV and (b) MNoV have high probability for mitochondrial localization. c, 3D structures of 4HB domain of NS3 (25-158aa) and mouse MLKL (1-125aa) derived from AlphaFold. d, Side views of the structure of mouse MLKL (1-125aa) (yellow), predicted by AlphaFold, was superimposed on mouse MLKL (green) structure previously reported26.
Extended Data Fig. 7 Expanded alignment of cellular and viral 4HB domain sequences.
a, Sequences of cellular MLKL 4HB domains were retrieved from NCBI. Accession numbers are present in Supplemental Table (1–3). Sequences were aligned using MUSCLE in Geneious Prime. Residues matching consensus are colored. b, NS3 N-terminus amino acid multiple sequence alignment (residues 1-158 relative to MNoV) for thirty-one calicivirus species including representatives from norovirus genogroups. Sequences were retrieved from NCBI. Viral species and strains were selected based on ICTV. Accession numbers are present in Supplemental Table (1–3). Sequences were aligned using MUSCLE in Geneious Prime. Residues matching consensus are colored.
Extended Data Fig. 8 Expanded alignment of cellular and calicivirus 4HB domain sequences.
Sequences were retrieved from NCBI. Viral species and strains were selected based on ICTV. Accession numbers are present in Supplemental Table. Sequences were aligned using MUSCLE in Geneious Prime. Residues matching consensus are colored.
Extended Data Fig. 9 MNoV infection and expression Full-length or N-terminal NS3 of MNoV altered mitochondria membrane potential and increased mitochondrial ROS abundance.
a, Representative confocal imaging of BV2 cells stably expressing inducible constructs of MNoVCR6 NS3-FL, NS3-N and NS3-C in the presence doxycycline for 10 h. Flag-tagged proteins (green) were detected by immunofluorescence with mitochondrial marker COX IV (red). Scale bar, 10 μm. b, Representative microscopy imaging of NS3 subcellular localization in HEK293T cells. C-terminal Flag-tagged NS3-FL, NS3-N and NS3-C transfected HEK293T cells, the distribution of NS3 (green) was detected by immunofluorescence with cellular membrane marker (yellow). Scale bar, 5 μm. (c–e) Full-length, N- or C-terminal of NS3 fused to a C-terminal Flag tag were stably expressed in BV2 cells under a tetracycline-inducible promoter. Doxycycline was used to induce NS3-FL, NS3-N and NS3-C expression. Cells were first gated off forward and side scatter. c, Flow cytometric analysis of mitochondrial membrane potential (Ψm) in the absence (black) or presence (red) of doxycycline measured by TMRM fluorescence. d, Flow cytometric analysis of mitochondrial status in the absence or presence doxycycline. Gates represent cells with damaged mitochondria. e, Flow cytometric analysis of mitochondrial ROS in the absence (black) or presence (red) of doxycycline. f, Flow cytometric analysis of mitochondrial status in macrophages infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. Gates represent cells with damaged mitochondria. g, Flow cytometric analysis of mitochondrial ROS in macrophages infected with MNoVCW3 and MNoVCR6 at a MOI of 5 for 12 h. Data in (a,b) are representative of two independent experiments. Data in (c–g) are representative of two independent experiments with three technical replicates of each condition.
Extended Data Fig. 10 Purification of recombinant MNoV NS3-FL and NS3-N, and evidence of self-association and formation of size-selective pores.
a–c, NS3-FL and NS3-N expressing His6-MBP tag were affinity purified by amylose resin. The His6-MBP tag was cleaved and the protein was further purified with Ni2+ resin, followed by size-exclusion chromatography of the purified NS3-FL (a) and NS3-N (b). The second peak of each was collected and Coomassie blue-stained gel (c) is shown. d, Thermal stability shifts measured NS3-FL and NS3-N after purification. e, Purified FL, N- or C-terminal NS3 proteins were incubated with PC liposomes. Liposome leakage was monitored by measuring DPA chelating-induced fluorescence of released Tb3+ relative to that of Triton X-100 treatment. f, Purified NS3-FL protein was incubated with varying sizes of FITC-dextran that was encapsulated in cardiolipin liposomes. Liposome leakage was measured as described in methods. g, Representative agar plates showing transformed E. coli colonies for NS3-FL, NS3-N and NS3-C. h, Bacterial colony-forming units (CFU) per transformation for NS3-FL, NS3-N and NS3-C are shown in the logarithmic form (log10). i, Flag immunoprecipitation of lysates of HEK293T cells, transfected with GFP-NS3-FL, GFP-NS3-N, GFP-NS3-C and/or Flag-NS3-FL, Flag-NS3-N, Flag-NS3-C, were analyzed by immunoblot with indicated antibodies. j, Flag immunoprecipitation of lysates of HEK293T cells, transfected with GFP-NS3-FL and/or Flag-NS3-FL, Flag-NS3-N, Flag-NS3-C, were analyzed by immunoblot with indicated antibodies. k, Oligomerization of NS3 on the liposome membrane. Full length MBP-NS3 proteins were incubated with cardiolipin or OMM liposomes and were subjected to BS3-mediated crosslinking followed by SDS-PAGE gel electrophoresis and Coomassie blue staining. Data in (a–e and g) representative of three independent experiments and in (i–k) representative of two independent experiments. Data in (f and h) are pooled from three independent experiments. Data are presented as mean ± s.d.
Extended Data Fig. 11 MNoV infection induces externalization of CL.
a, BV2 cells were left untreated, treated with 2 μM staurosporine (STS) for 4 h, or infected with MNoVCW3 and MNoVCR6 (MOI = 1) for 6 h. Mitochondria were isolated and stained with Annexin V-Alexa Fluor 647 and analyzed by flow cytometry. b, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI = 0.1) infected WT and Pls3 siRNA knockdown BMDMs cells for 12 h. c, Pls3 was deleted from BV2 cells by CRISPR genome editing. Lysates of WT or knockout cells were blotted with anti-PLS3 and anti-actin antibodies. d, Representative images of MNoVCW3 and MNoVCR6 (MOI = 0.1) infected WT and Pls3−/− cells for 12 h. Propidium iodide (PI) was added to the cells 20 min before imaging. Scale bar, 100 μm. e, Comparison of ATP levels in MNoVCW3 and MNoVCR6 (MOI = 0.1) infected WT and Pls3−/− cells for 12 h. f, WT and Pls3−/− BV2 cells infected with MNoVCR6 at a MOI = 0.1 for 12 h and supernatant virus was measured by plaque assay. Data in (a,c and d) representative of two independent experiments. Data in (b,e and f) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d. Data in (f) is analyzed by two-tailed Student’s t-test.
Extended Data Fig. 12 Mitochondrial localization of NS3 is essential for inducing cell death and norovirus infection.
a, Representative confocal microscopy images of staining for dsRNA (green) and DAPI (blue) in BMDMs infected with WT (MNoVCW3 and MNoVCR6) or mutant (MNoVCW3ΔN and MNoVCR6ΔN) virus at MOI of 5 for 12 h. Scale bar, 5 μm. b, BMDMs derived from wild-type mice were infected with WT (MNoVCW3 and MNoVCR6) or mutant (MNoVCW3ΔN and MNoVCR6ΔN) viruses. Cells collected at indicated time points and total cell lysates were subjected to anti-NS1 or anti-actin immunoblotting. c, BV2 cells infected with WT (MNoVCR6) and mutant (MNoVCR6ΔN) MNoV at MOI = 1 and viral genomes that were in the supernatant were quantified by qPCR. d, Representative images of Sytox-Green-stained BV2 cells infected with WT (MNoVCR6 and MNoVCW3) and mutant (MNoVCR6ΔN20 and MNoVCW3ΔN20). Scale bar, 100 μm. (e–h) WT mice were challenged with 106 PFU of MNoVCR6 or MNoVCR6ΔN20 perorally. All mice survived infection. Viral genomes were quantified in the (e) MLN, (f) colon, (g) ileum, (h) feces at 7 days post infection. N = 5 for both groups. Dashed line represents the limit of detection. (i) Survival of Stat1−/− mice after challenge with 106 PFU of MNoVCW3 (n = 6) and MNoVCW3ΔN20 (n = 6). Data in (a,b and d) are representative of two independent experiments. Data in (c) pooled from three independent experiments. Data in (e–h) pooled from two independent experiments. Data are presented as mean of ± s.d. Statistical analysis was conducted using two-tailed Student’s t-test. P-value for (i) was calculated with a log-rank (Mantel-Cox) test.
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Wang, G., Zhang, D., Orchard, R.C. et al. Norovirus MLKL-like protein initiates cell death to induce viral egress. Nature 616, 152–158 (2023). https://doi.org/10.1038/s41586-023-05851-w
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DOI: https://doi.org/10.1038/s41586-023-05851-w
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