A viral necrosome mediates direct RIPK3 activation to promote inflammatory necroptosis

Abstract

Necroptosis is an inflammatory programmed cell death pathway triggered by RIPK3 activation through one of the upstream RHIM-domain-containing proteins including RIPK1, TRIF, and ZBP1. Whether necroptosis can be activated independent of the upstream signaling pathways leading to inflammatory pathogenesis remains ambiguous. Here, we revealed a mechanism in which a viral protein mediates direct RIPK3 activation resulting in severe inflammatory pathogenesis in patients. The nonstructural protein NSs of a pathogenic hemorrhagic virus, SFTSV, interacts with the RIPK3 kinase domain and forms biocondensate to promote RIPK3 autophosphorylation and necroptosis activation in an RHIM-independent manner. In parallel, sequestration of RIPK3 within the NSs-RIPK3 condensate inhibited RIPK3-mediated apoptosis and promoted viral replication. Infection with an SFTSV NSs mutant virus not forming NSs condensate triggered pronounced apoptosis resulting in reduced viral replication and decreased fatality in vivo. Blocking SFTSV-triggered necroptosis through depletion of MLKL or treatment with a RIPK3-kinase inhibitor reduced viral inflammatory pathogenesis and fatality in vivo. In contrast, blocking SFTSV-triggered apoptosis through depletion of RIPK3 resulted in enhanced viral replication and increased fatality in vivo. The virus-triggered necroptosis correlated with severe inflammatory pathogenesis and lethality in virus-infected patients. The NSs-RIPK3 condensate may represent a necroptosis activation mechanism that promotes viral pathogenesis.

Keywords: NSs; RIPK3; cell death; inflammatory pathogenesis; viral necrosome.

Fig. 1.

SFTSV infection triggers necroptosis in patient samples and multiple cell models. (A) IL-1α, IL-33, and mtDNA levels in the serum samples of SFTSV patients (survival and deceased) and healthy controls. (B and C) PBMCs isolated from healthy donors were infected with SFTSV at an MOI of 10 for 12, 24, 36, or 48 h. Cell death was determined by the LDH Cytotoxicity Assay Kit (B). Intracellular pMLKL, MLKL, pRIPK3, RIPK3, or NP were measured by western blot analysis (C). (D and E) THP-1^PMA cells were infected with SFTSV at an MOI of 10 for 6, 12, 18, or 24 h. Cell death was determined by the LDH Cytotoxicity Assay Kit (D). Intracellular pMLKL, MLKL, pRIPK3, RIPK3, or NP were measured by western blot analysis (E). Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA (A). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Necroptosis drives viral pathogenesis and lethality during SFTSV infection in vivo. (A and B) Mlkl^−/− or WT C57BL/6J mice (n = 15) were pretreated with anti-IFNAR1 monoclonal antibody and intraperitoneally infected with SFTSV (1 × 10⁴ FFU). Mice were monitored (A) and weighed (B) for 14 d. (C–J) Mlkl^−/− or WT C57BL/6J mice (n = 8) pretreated with anti-IFNAR1 monoclonal antibody were intraperitoneally infected with SFTSV (1 × 10⁴ FFU). Uninfected WT (n = 4) and Mlkl^−/− mice (n = 4) were served as control. Blood, liver, and spleen samples were harvested at 4 dpi. Viral loads in spleen and liver samples were quantified by qRT-PCR (C). Viral titers in spleen and liver samples were measured by focus-forming assay (D). Spleen sample of SFTSV-infected Mlkl^−/− or WT mice were subjected to RNA-seq. KEGG (E) and GO (F) enrichment analysis of significantly downregulated genes in the spleen sample of SFTSV-infected Mlkl^−/− mice compared with WT mice. Relative mRNA level of Cd14 and Mmp8 in spleen and liver samples was analyzed with qRT-PCR and normalized to uninfected WT controls (G). The histopathological changes in the liver and spleen were evaluated by H&E staining (H). (Scale bar, 100 μm.) Lymphocytes in blood samples were determined and calculated (I). IL-6 and IL-1β levels in the serum were measured by ELISA (J). (K) C57BL/6J mice were divided into four groups: SFTSV infection treated with the vehicle group (n = 8, red line), SFTSV infection treated with the Zharp-99 group (n = 8, green line), and mock infection treated with the vehicle group (n = 4), and mock infection treated with the Zharp-99 group (n = 4). Mice were pretreated with anti-IFNAR1 monoclonal antibody and intraperitoneally infected with 3 × 10⁴ FFU of SFTSV or the same volume of DMEM. Zharp-99 was given by intraperitoneal injection (2 mg/kg) twice a day for 5 d. Mice were monitored and weighed for 14 d. Data are shown as mean ± SEM. Statistical analyses were performed using the log-rank (Mantel–Cox) test (A and K), two-tailed unpaired t test, or Mann–Whitney test (C, D, G, I, and J). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.

Fig. 3.

NSs protein promotes necroptosis through facilitating RIPK3 phosphorylation in an RHIM-independent manner. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged RIPK3 and Strep-tagged viral proteins. Cell lysates were harvested at 24 h posttransfection and subjected to Flag immunoprecipitation, followed by western blot analysis with anti-Flag antibody for RIPK3, anti-Strep antibody for viral proteins except for polymerase (Lp). Lp was detected with homemade rabbit antibody against Lp. (B) Hela^RIPK3-TO cells were treated with Dox (50 ng/mL) and infected with SFTSV (MOI = 5) for 24 h, followed by staining with antibodies against RIPK3 (green) and NSs (red). Nuclei were stained by DAPI (blue). (Scale bar, 5 μm.) (C) Quantitative analysis of the colocalization between RIPK3 and NSs was performed with ImageJ. (D) RIPK3 was truncated into the N-terminal kinase domain and C-terminal proline-rich/RHIM-containing domain. (E) HEK293T cells were cotransfected with plasmids expressing Flag-tagged NSs and Strep-tagged full-length or truncated RIPK3 proteins. Cell lysates were harvested at 24 h posttransfection and subjected to Flag immunoprecipitation, followed by western blot analysis with anti-Flag antibody for NSs and anti-Strep antibody for full-length or truncated RIPK3. * Unspecific band.

Fig. 4.

NSs forms biocondensate to promote RIPK3 autophosphorylation. (A) HEK293T cells transfected with plasmid expressing eGFP-NSs were treated with 3.5% 1.6-HD or vehicle for 30 min, and fixed for fluorescence microscopy. Nuclei were stained by DAPI (blue). (Scale bar, 5 μm.) (B) FRAP quantification of eGFP-NSs condensates over a period of 200 s. (C) Representative micrographs of eGFP-NSs condensates before and after photobleaching. (Scale bar, 5 μm.) (D) Fusion of eGFP-NSs condensates in a HEK293T cell. (Scale bar, 5 μm.) (E and F) HEK293T cells were cotransfected with plasmids expressing Flag-tagged RIPK3 and Strep-tagged NSs^WT or NSs^{N122A–S123A} mutant. Cell lysates were harvested at 24 h and subjected to Strep pulldown (E) or Flag immunoprecipitation (F) followed by western blot analysis with indicated antibodies. (G) Hela cells were cotransfected with plasmids expressing Flag-tagged RIPK3 and Strep-tagged NSs^WT or NSs^{N122A–S123A} mutant. Cell lysates were harvested at 24 h for western blot analysis with indicated antibodies. (H) Hela cells were cotransfected with plasmids expressing Strep-tagged RIPK3 kinase domain (RIPK3^1–356) and Flag-tagged NSs^WT or NSs^{N122A–S123A} mutant. Cell lysates were harvested at 24 h for western blot analysis with indicated antibodies. * Unspecific band.

Fig. 5.

Modulation of cell death pathways by NSs condensation determines SFTSV pathogenesis in vivo. (A and B) C57BL/6J mice (n = 12) were pretreated with anti-IFNAR1 monoclonal antibody and intraperitoneally infected with SFTSV-NSs^WT or SFTSV-NSs^{N122A–S123A} (1 × 10⁴ FFU). Mice were monitored (A) and weighed (B) for 14 d. (C–E) C57BL/6J mice pretreated with anti-IFNAR1 monoclonal antibody were intraperitoneally inoculated with SFTSV-NSs^WT or SFTSV-NSs^{N122A–S123A} (1 × 10⁴ FFU, n = 5). Uninfected mice (n = 4) were served as control. Viral loads in serum, spleen, and liver samples harvested at 4 dpi were quantified by qRT-PCR (C). Viral titers in serum, spleen, and liver samples were measured by focus-forming assay (D). Spleen samples were harvested at 2 dpi, TUNEL staining representing activation of apoptosis (E, Upper panels). (Scale bar, 100 μm.) Immunostaining of pMLKL representing activation of necroptosis (E, Lower panels). (Scale bar, 50 μm.) Data are shown as mean ± SEM. Statistical analyses were performed using the log-rank (Mantel–Cox) test (A), two-tailed unpaired t test, or Mann–Whitney test (C and D). *P < 0.05; **P < 0.01.