Intracellular sensing of complement C3 activates cell autonomous immunity

Abstract

Pathogens traverse multiple barriers during infection, including cell membranes. We found that during this transition, pathogens carried covalently attached complement C3 into the cell, triggering immediate signaling and effector responses. Sensing of C3 in the cytosol activated mitochondrial antiviral signaling (MAVS)-dependent signaling cascades and induced proinflammatory cytokine secretion. C3 also flagged viruses for rapid proteasomal degradation, preventing their replication. This system could detect both viral and bacterial pathogens but was antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral rupintrivir inhibited 3C protease and prevented C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol.

Figure 1

Complement C3-bound virus induces NF-κB signaling in non-immune cells. (A) NF-κB activity of HEK293T cells challenged with PBS, AdV, serum, heat-inactivated serum (HI Serum), EGTA-treated serum (Serum+EGTA), Plasmin-treated serum (Serum+Plasmin), or AdV incubated with the previous sera. (B) HEK293T cells treated with AdV incubated with Serum, HI Serum, antibody-depleted Serum (Serum-Ig), or HI Antibody-depleted Serum (HI Serum-Ig). (C) Concentration of serum components IgM, IgA, IgG, IgD, IgE, C3, C4, Mannan-Binding Lectin (MBL), C-Reactive Protein (CRP) and Pentaxin 3 (PTX3) bound to AdV after incubation of AdV+Serum as measured by ELISA. (D-F) NF-κB activity of HEK293T cells treated with AdV incubated with (D) serum deficient in complement components (Serum-C1–C6), (E) serum deficient in complement C4 (AdV+Serum-C4) or heat inactivated Serum-C4 (AdV+HI Serum-C4) or (F) cobra venom factor treated serum (AdV+Serum+CVF). (G) NF-κB activity of HEK293T cells follwing challenge with AdV+Serum, AdV+HI Serum or Serum-C3 reconstituted with purified C3 (Serum-C3 + C3). (H) HEK293T treated with AdV incubated with serum deficient in factor B (AdV+Serum-fB) or factor D (AdV+Serum-fD). (I) NF-κB activity of HEK293T cells treated with AdV incubated with C3-Factor B-Factor D (AdV+C3fBfD) with and without subsequent virus pelleting. Data are representative of three experiments; results in (A,B, D–I) as fold change over PBS treated controls, mean +/− SEM, N=6; data in (C), mean +/− SEM, N=3.

Figure 2

Signaling in response to C3-bound virus is mediated by an intracellular receptor. (A) Immunoblot for complement receptors and GAPDH (loading control) in HEK293T, HeLa, Caco-2, Normal Human Lung Fibroblasts (NHLF) and THP-1 monocyte cells. (B) HEK293T cells treated with control, CD46 or CD55 siRNA, challenged with AdV+Serum, AdV+HI Serum and AdV+Serum+CVF. NF-κB activity after treatment with DMSO, Bafilomycin A1 (BafA1), or CID1067700 (CID), challenged with AdV+C3fBfD, or TNF in HEK293T (C), NHLF cells (D) or THP-1 cells (E). (F) Endosomal disruption in HEK293T measured by delivery of a nano-luciferase expressing plasmid present in cell supernatant by infecting AdV, after treatment with DMSO, BafA1 or Rab7, as relative luminescence units (RLU). (G) Confocal microscopy of HeLa cells 30 minutes after treatment with AdV with AlexaFluor-488 labeled C3, stained with DAPI and antibody to AdV. Scale bars, 20μm. (H) HEK293T cells treated with Beads incubated with C3fBfD (Beads+C3fBfD), with or without transfection reagent. Data are representative of three experiments; results in (B–E, H) as fold change over PBS treated controls, mean +/− SEM, N=6; data in (F), mean +/− SEM, N=3.

Figure 3

C3-mediated signaling initiates proinflammatory cytokine production. (A) NF-κB luciferase activity in NHLF cells treated with DMSO, 5Z-7-Oxozaeanol, IKK VII, or Panepoxydone. (B) Levels of total and phosphorylated IKKα, IκB, p65 in HEK293T cells treated with AdV+C3fBfD 4h post-infection as measured by ELISA. Levels of (C) NF-κB components (D) IRF family proteins and (E) AP-1 components measured by DNA binding ELISA from NHLFs 4h post-challenge with AdV+C3fBfD. Levels of cytokines 24 h after challenge by AdV incubated with (F) Serum, HI Serum, or Serum+CVF or (G) AdV+C3fBfD. Data are representative of three experiments; results in (A–E) as fold change over PBS treated controls, mean +/− SEM, N=3; data in (F, G), mean +/− SEM, N=3.

Figure 4

C3 promotes intracellular restriction of virus. (A-D) Levels of infection in HeLa cells after challenge with GFP-encoding adenovirus AdV, pre-treated as indicated. (E) Levels of infection in HEK293T cells treated with control, CD46 or CD55 siRNA. Levels of infection in HeLa cells treated with (F) DMSO, or 3-MA, KU55933 or Gö6976 or (G) control or TRIM21-directed siRNA. HeLa cells after stimulation with (H) BSA, or IFN-α or (I) treated with DMSO, or VCP inhibitor DBeQ or proteasome inhibitor epoxomicin. (J) Immunoblot for AdV capsid component hexon and GAPDH in HeLa cells treated with DMSO or epoxomicin at indicated times after challenge with AdV+C3fBfD. (K) Levels of infection in NHLF cells treated with DMSO or epoxomicin. Data are representative of three experiments; results in (A–I, K) percentage infected cells normalized to AdV only controls, mean +/− SEM, N=3.

Figure 5

C3 detection is conserved in mammals and is active against RNA and DNA non-enveloped viruses and bacteria. (A) HEK293T cells challenged with AdV incubated with human, mouse, cat, rabbit and guinea pig serum. (B) HEK293T (human), Vero (African green monkey), MEF (mouse), FEA (cat) and MDCK II (dog) cells challenged with AdV+Serum or AdV+HI Serum. (C) NF-κB activity after challenge with wild-type adenovirus 5 (WT AdV), human papillomavirus virus like particles (HPV), human astrovirus-1 (hAstV), feline calicivirus (FCV), human rhinovirus 14 (HRV), poliovirus 2 (PV), and coxsackievirus B3 (CVB). WT AdV and HRV carried out on HEK293T; HPV, PV and CVB on HeLa; hAstV on Caco-2; and FCV on FEA cells. (D) NF-κB activity in HeLa after challenge with RSV incubated with sera. (E) NF-κB activity in HeLa, Caco-2 and MEF cells after challenge with WT Salmonella+C3fBfD. (F) MEF cells challenged with ΔSif Salmonella+C3fBfD. Results are representative of three experiments, as fold change over PBS treated controls; data in (A–D), mean +/− SEM, N=6; data in (E, F), mean +/− SEM, N=3.

Figure 6

Viral antagonism of C3-mediated signaling. NF-κB induction by the heat-labile component of serum activity following infection of HEK293T cells by AdV, HPV, hAstV, PV, HRV and UV-inactivated HRV (UV-HRV), shown as fold change of Virus+Serum over Virus+HI Serum signalling levels. Immunoblot for C3 (B) and AdV (C) in samples comprising C3 deposited on AdV incubated with recombinant HRV 3C Protease (Ext HRV 3C) or components thereof. (D) NF-κB activity in HEK293T expressing HRV 3C Protease (HRV 3C Pro) or PV 3C Protease (PV 3C Pro). (E) NF-κB activity in HEK293T treated with Bovine Serum Albumin (BSA), Ext HRV 3C or recombinant PV 3C Protease (Ext PV 3C), with AdV complexes pelleted. (F) Levels of infection of HeLa cells challenged with AdV, AdV+Serum, AdV+HI Serum, and AdV+Serum+CVF treated with Ext HRV 3C, or Ext PV 3C, followed by pelleting. (G) Immunoblot for C3 after infection of HeLa cells expressing empty vector or HRV 3C Pro by AdV+C3fBfD or HRV+C3fBfD. (H) NF-κB activity in HEK293T cells treated with DMSO or 3C antagonist rupintrivir, challenged with HRV incubated with sera. (I) Immunoblot for C3 after infection of HeLa with AdV+C3fBfD or HRV+C3fBfD treated with DMSO or rupintrivir. (J) NF-κB activity induced by the heat-labile component of serum upon AdV, HRV or PV infection of HEK293T cells treated with DMSO or rupintrivir at indicated times post-infection. (K) NF-κB activity in THP-1 cells treated with DMSO or rupintrivir and challenged with sera-incubated HRV. (L) IFN-β ELISA from NHLF cells infected with HRV under different conditions after treatment with DMSO, Ext HRV 3C or rupintrivir. Data from dot plots are from five experiments (dots are mean of N=6), bar graphs are representative of three experiments, data in (D, E, H, K) as mean +/− SEM N=6, data in (L), mean +/− SEM N=4, data in (J), mean +/− SEM, N=3.

Figure 7

C3-mediated signaling is MAVS dependent. (A) NF-κB activity in HEK293T cells treated with control peptides, or inhibitors of MyD88 or TRIF after challenged with AdV+C3fBfD, or LPS. (B-F) NF-κB activity following challenge by AdV incubated as indicated on HEK293T cells treated with (B) control, RIG-I or MDA5 siRNA, (C) DMSO, or inhibitors Syk I and Syk III, (D) control, MAVS or STING-directed siRNA, (E) control, TRAF2, TRAF3, TRAF5, TRAF6 or pooled TRAF2,3,5,6 (siT2,3,5,6) siRNA and (F) control, TRAF6 or p62 siRNA. (G) IRF3, 5, and 7 binding to consensus DNA response elements in HEK293T cells treated with siRNA as in E. (H) IRF3, 5, and 7 binding to DNA response elements in HEK293T cells treated with control, MAVS or TBK1 specific siRNAs, or with inhibitor, BX795. Data are representative of three experiments; results in (A–H) as fold change over PBS treated controls, mean +/− SEM, N=6.

Figure 8

C3 leads to concurrent and independent signaling and restriction. (A) NF-κB activity in HEK293T cells treated with control, or MAVS and TRIM21 siRNA. (B) Levels of infection of HeLa cells treated with control, or MAVS and TRIM21 siRNA, then DMSO or epoxomicin, after infection with AdV, AdV+Serum or AdV+HI Serum. (C) Cells as in B, infected with AdV, or AdV+C3fBfD. (D) NF-κB activity in HEK293T cells treated with control, or MAVS and TRAF6 siRNA, transfected with Beads+C3fBfD. (E) IRF3, 5, and 7 binding to DNA response elements in HEK293T cells treated with DMSO or epoxomicin. (F) Levels of infection of HeLa cells treated with DMSO or epoxomicin and panepoxydone after challenge with AdV treated as indicated. (G) Relative level of Sindbis infection of HeLa cells in the presence of fresh medium (DMEM), medium with IFN-α, or supernatant from cells treated with control or MAVS siRNA, challenged with PBS, or AdV and C3fBfD. (H) Viability of HeLa cells challenged with dilutions of C3fBfD in a spreading HRV infection assay. Cells treated with control or MAVS siRNA, then with DMSO, MG132 or panepoxydone. Data are representative of three experiments; results in (A–G) mean +/− SEM, N=3; data in (H), mean +/− SEM, N=5.