Intracellular sensing of complement C3 activates cell autonomous immunity

Abstract

Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol.

Host colonization by intracellular pathogens requires penetration of mucosal layers and dissemination through extracellular fluids. Humoral immunity has two major components - the heat-labile complement system and the heat-stable immunoglobulin system, and provides robust protection against invading pathogens. Whilst such protection has been extensively studied, insights into its function have recently emerged. Antibodies have been shown to be carried into cells by non-enveloped viruses during infection, where they act as a danger associated molecular patterns (DAMPs) to activate innate immunity(1, 2) and inhibit viral replication both in vitro(2-5) and in vivo(6). We hypothesized that this phenomenon may not be unique to antibodies and that mislocalization of serum proteins as a result of pathogen movement from extracellular to intracellular compartments might be a strategy widely exploited by host immunity.

The complement system is composed of more than thirty proteins(7), with three activation methods – classical (antibody-directed), lectin (mannan-binding lectin or ficolin-directed) and alternative (spontaneous) pathways, leading to covalent deposition of C3 on the pathogen surface. Effector functions of complement stem from this deposition: C3 prevents receptor engagement, acts as an opsonin and activates the terminal complement components to form a membrane attack complex, while cleaved components are anaphylatoxins. Most antiviral complement studies have been with enveloped viruses(8). However, non-enveloped viruses and adenoviral gene therapy vectors are also susceptible to complement deposition(9) while some possess specific complement evasion strategies(10, 11). Due to the lack of a lipid bilayer, the membrane attack complex cannot form on non-enveloped viruses, so aside from blocking receptor engagement, it is unknown how complement inhibits their infection.

Results

Complement Component C3 elicits NF-κB activation

Antibody in the cytosol has been shown to activate innate immune signaling cascades, establishing an anti-viral state via receptor, TRIM21(1, 2). We tested whether other serum components are able to elicit similar responses by attaching to incoming pathogens. Infection of HEK293T cells carrying a NF-κB-driven luciferase reporter, with an adenovirus type 5 vector (AdV) did not activate NF-κB (Figure 1A). In contrast, incubation of AdV with normal human serum (Serum) led to robust NF-κB activation. Treatment of this serum by heat-inactivation at 56°C (HI Serum), or addition of EGTA (Serum+EGTA) to chelate calcium, both lead to a reduction in NF-κB induction. Plasmin is a host serine protease that is pathogenically activated by Staphylococci to cleave protein components from their capsule for immune evasion(12). Serum was treated with plasmin for 30 minutes, before quenching with alpha-2-antiplasmin (Serum+Plasmin), and then incubated with AdV (Figure 1A). Plasmin treatment abolished NF-κB induction but did not alter AdV infection. We then determined the antibody component of signaling, with the use of antibody-depleted serum (Serum-Ig), and a heat-inactivated form (HI Serum-Ig). Incubation of AdV with either antibody-depleted serum (Serum-Ig) or a heat-inactivated form (HI Serum-Ig) confirmed that antibody is responsible for part of this NF-κB activation(1). However, a significant heat labile component functioned independent of antibody (Figure 1B). This signaling component within serum consisted of a heat-labile, calcium-dependent protein (Figure 1A-B) which appears to function alongside and independent of antibody – consistent with known properties of the complement system(7). To categorize which serum components can attach to non-enveloped virus particles and thereby activate signaling, serum was incubated with AdV, spun through 30% sucrose to remove unbound proteins, with attached proteins detected by ELISA (Figure 1C). Antibodies of IgM, IgA and IgG isotypes bound to AdV, but IgD and IgE did not. Complement component C3 was strongly detected, showing that it deposited on AdV. C4 was also detected, indicating that classical activation had occurred. Mannan-binding lectin (MBL), and C-reactive protein (CRP) were not detected, suggesting that the lectin pathway was not required for complement deposition on AdV. Pentaxin 3 (PTX3) has antiviral activity against influenza(13) but was not detected bound to AdV (Figure 1C). These data are supportive of complement activating NF-κB upon AdV infection. Infection experiments were carried out in the presence of serum lacking specific complement components (Figure 1D). Whilst there was reduced NF-κB activation in C1 and C2-deficient serum (Serum-C1 and Serum-C2 respectively), only C3-deficient serum (Serum-C3) diminished induction comparably with heat inactivation. Serum deficient in components after C3, Serum-C5 and Serum-C6, did not have any impact compared with AdV+Serum. C4-deficient serum (Serum-C4) also gave similar responses to that observed from Serum-C1 and Serum-C2 (Figure 1E). To further confirm the importance of C3, Serum was treated with Naja naja kaouthia cobra venom, which is known to cleave and inactivate C3(14), and this resulted in a similar reduction in NF-κB induction by serum as heat-inactivation (Figure 1F). NF-κB activation in C3-deficient serum was restored by the addition of recombinant C3 protein (Figure 1G). Serum deficient in factor B or factor D, components of the alternate pathway(7), gave similarly diminished NF-κB activation to serum lacking C1- or C2, suggesting that both the alternate and classical pathways are important for C3 deposition (Figure 1D, H). We reconstituted the alternate deposition pathway using purified proteins (AdV+C3fBfD). Activated purified C3 potently induced NF-κB in the presence but not the absence of AdV (Figure 1H). To test whether signaling was mediated by C3 attached to virus or by cleaved anaphylatoxins, AdV+C3fBfD was pelleted through 30% sucrose. Pelleted AdV+C3fBfD stimulated NF-κB comparably with unpelleted material showing that it was C3-bound AdV that initiated signaling (Figure 1I). Together, these data show that C3 attached to the pathogen surface activates NF-κB upon infection.

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.

Non-immune cells have an intracellular C3 Receptor

Most cells express inhibitory complement receptors CD46 (also known as Complement Regulatory Protein) and CD55 (Decay Accelerating Factor), while professional immune cells express a number of activating complement receptors(15). Consistent with this, HEK293T, HeLa, and Caco-2 cells, and primary normal human lung fibroblasts (NHLF) all expressed CD46 and CD55 (Figure 2A); whilst only THP-1 monocytes expressed the activating receptors, complement receptor 1 (CR1, also known as CD35), CR3 (made up of CD11b and CD18) and CR4 (CD11c and CD18). Depletion of CD46 or CD55 (Figure S1A) had no impact on signaling detected in HEK293T cells (Figure 2B), suggesting that C3-mediated NF-κB activation is not due to detection by these cell surface receptors.

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.

Antibodies activate immune signaling in non-professional cells when carried into the cytosol by an infecting virus(1). We investigated whether C3 similarly activates signaling when carried into the cell during virus infection. As AdV infection is dependent upon receptor-mediated endocytosis(16), we tested whether endocytosis inhibitors prevent C3 activation of NF-κB. Inhibition of endosomal acidification by Bafilomycin A1 (BafA1), or the endocytic pathway by Ras superfamily inhibitor CID1067700 (CID)(17), abolished AdV+C3fBfD mediated NF-κB signaling in HEK293T, whilst induction by Tumor Necrosis Factor (TNF) remained unaffected (Figure 2C). The same effect was seen in NHLFs (Figure 2D). However signaling in THP-1 cells, which have activatory cell surface receptors for C3, was not affected by endocytosis inhibitors (Figure 2E). To confirm that C3 induced signaling in non-professional cells is a result of intracellular sensing, an endosomal disruption assay was used, in which cytosolic delivery of a constitutively-expressing nano-luciferase plasmid was measured. Incubation of cells with C3fBfD had little impact on AdV escape from endosomes, while BafA1 and CID abolished it (Figure 2F), verifying that AdV does not reach the cytosol in the presence of these inhibitors. To test whether C3 remains attached to AdV during entry of the virus, cells were examined by confocal microscopy. AdV particles could be detected within the cell and co-localized with AlexaFluor-488 labeled C3 deposited prior to infection (Figure 2G). Moreover, no C3 was detected in the absence of virus suggesting that C3 is deposited onto virus and transported into the cell. Finally, to show that signaling by intracellular C3 is independent of co-recognition of viral pathogen associated molecular patterns (PAMPs), beads were incubated with the C3fBfD mix, and transfected into cells (Figure 2H). Only Beads+C3fBfD that had been transfected into cells were capable of signaling. Together, these data suggest that non-immune cells possess a signaling pathway that allows them to sense intracellular complement C3.

C3 signaling leads to pro-inflammatory cytokine production by activating NF-κB, IRF and AP-1 transcription factors

Next, we investigated which signalling pathways are activated by intracellular C3 sensing. Canonical NF-κB signal transduction involves the TAB-TAK complex, which activates the IKK complex to phosphorylate IκB, promoting IκB degradation and releasing NF-κB to translocate into the nucleus. Inhibitors of the TAB-TAK complex (5Z-7-oxozaeanol), the IKK complex (IKK VII) and NF-κB release (panepoxydone) were each sufficient to inhibit signaling (Figure 3A). Furthermore, increased phosphorylation of these components was detected upon infection with virus carrying deposited C3 (Figure 3B). NF-κB components p65, p50 and p52 were activated by AdV+C3fBfD (Figure 3C), as well as IRF3, 5 and 7 (Figure 3D) and the AP-1 family, c-Jun, JunB, JunD and FosB (Figure 3E). Notably, c-Fos was activated by virus regardless of C3, suggesting that other mechanisms are involved in its activation. Together, these data indicate that C3 is sensed by a Pattern Recognition Receptor (PRR) that activates classical immune transduction pathways. Importantly, immune activation by complement-coated virus but not virus alone was sufficient to induce secretion of pro-inflammatory cytokines, as demonstrated by the detection of IL-6, TNF, CCL4, IL-1β and IFN-β by ELISA after challenge with AdV+Serum and AdV+HI Serum (Figure 3F), as well as the reconstituted alternate pathway of AdV+C3fBfD (Figure 3G).

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.

C3 enables proteasome-dependent restriction of virus infection

The above data suggest that C3 can act as a DAMP to activate innate immunity. Next, we investigated whether C3, also mediates a direct effector response to inhibit viral infection. We used an adenovirus vector that expresses GFP after productive infection. AdV incubated with Serum, HI Serum, Serum+EGTA, or Serum+Plasmin and AdV alone was added to HeLa cells, with infected cells enumerated by flow cytometry (Figure 4A). As with signaling, restriction by a heat-labile, calcium-dependent, protein component capable of functioning independently of antibody was observed (Figure 4B). This restriction was C3-dependent, as shown by the loss of heat-labile neutralization following CVF treatment (Figure 4C). Moreover, restriction could be reconstituted in the absence of serum using C3fBfD (Figure 4D). Depletion of CD46 or CD55 had no effect on restriction, showing that capture via these membrane complement receptors is not responsible for this phenotype (Figure 4E, S1A). Binding to complement receptor CD46 has been suggested to stimulate autophagy(18), however autophagy inhibitor 3-methyladenine (3-MA), and phosphatidylionsitol-3-kinase inhibitors KU55933 and Gö6976(19) had no effect on restriction (Figure 4F), but induced p62 retention (Figure S1B). TRIM21 is required for intracellular neutralization of antibody-coated pathogens(4, 5). Depletion of TRIM21 removed the heat stable component of restriction, but did not affect the heat labile component (Figure 4G, S1C), confirming that TRIM21 has a role in antibody-mediated neutralization but not complement-mediated restriction.

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.

A characteristic of antiviral immunity is interferon regulation and most antiviral genes are interferon stimulated(20). TRIM21 is an interferon stimulated gene(4, 5) and the efficiency of antibody-dependent intracellular neutralization is dependent upon the amount of TRIM21 in the cell. To determine whether C3-mediated restriction was also interferon inducible, cells were stimulated prior to infection (Figure 4H). We found that interferon increased the ability of the cell to restrict viral infection via complement C3, suggesting that complement recognition occurs through an interferon stimulated gene.

Antibodies mediate intracellular neutralization by targeting viruses for degradation by the AAA-ATPase VCP (also known as p97)(3) and the proteasome(4). To determine whether C3 also mediates restriction by recruiting these enzymes, we tested the effect of DBeQ (a VCP inhibitor) and epoxomicin (a proteasome inhibitor). Either inhibitor was sufficient to perturb restriction of AdV by C3, suggesting that C3 activates an intracellular, VCP and proteasome-dependent pathway (Figure 4I). To directly test whether the block to infection was the result of targeted degradation of incoming virions, we performed a fate-of-capsid experiment in which levels of the AdV major capsid protein, hexon, were measured at different time points. Hexon was rapidly degraded in a proteasome-dependent manner but only when complement was present (Figure 4J). These restriction phenotypes did not just occur in HeLa cells, but could also be replicated in primary NHLF cells (Figure 4K). Thus, in addition to activating innate immunity, the attachment of C3 to invading virions labels them for degradation by VCP and the proteasome, thereby restricting virus infection.

Intracellular complement immunity is conserved in mammals

Complement protein/receptor interactions are thought to be species specific(21), although recent observations have disagreed with this view(22). We investigated whether intracellular complement immunity is conserved amongst mammals. Serum from different mammalian species was incubated with AdV, pelleted to remove unattached complement components and then added onto HEK293T cells. Human, mouse, cat, rabbit and guinea pig serum all elicited complement-mediated signaling (Figure 5A). We then tested whether this signaling was present in cell lines derived from different mammals. Human (HEK293T), African green monkey (Vero), mouse (mouse embryonic fibroblasts, MEF), cat (feline embryonic airway, FEA) and dog (Madin-Darby canine kidney, MDCK) cells all signaled in response to complement-coated AdV (Figure 5B). These data suggest that a system of intracellular complement immunity is conserved within mammals.

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.

Intracellular complement immunity is effective against diverse pathogens

As complement sensing was independent of viral PAMPs (Figure 2H), we hypothesized that it may be an effective way of activating immunity during infection by diverse pathogens. Complement sensing allowed detection of infection by replication-competent wild-type adenovirus 5 (WT AdV), confirming previous experiments carried out with replication deficient virus lacking the E1 and E3 genes (Figure 5C). In addition, infection by human papillomavirus virus-like particles (HPV), human astrovirus 1 (hAstV), feline calicivirus (FCV), human rhinovirus 14 (HRV), poliovirus 2 (PV) and coxsackievirus B3 (CVB) also elicited NF-κB induction in a complement-dependent manner (Figure 5C). These viruses utilize different strategies for cell entry. Adenovirus(23) and rhinovirus 14(24) lyse the endosome during infection, whilst poliovirus(25) and coxsackievirus(26) form a pore in the endosome membrane. Accordingly, endosomal disruption assays revealed a correlation between endosomal disruption and the potency of complement mediated signaling for these viruses (Figure S1K).

Complement deposition on an enveloped virus occurs on the lipid membrane and not its internal capsid. Consequently, during infection by an enveloped virus, complement should be left behind on the outside of the plasma membrane or inside endosomes. To test this, we infected cells with respiratory syncytial virus (RSV), an enveloped virus that enters through membrane fusion(27), in the presence of serum. As expected, no complement signaling was observed during RSV infection (Figure 5D).

Non-enveloped viruses are not the only intracellular pathogen capable of being targeted by complement deposition. Salmonella enterica enterica serovar Typhimurium can escape from their salmonella containing vesicles, and replicate in the cytosol(28). Antibody deposited on bacterial surfaces has previously been shown to be detected by TRIM21(1). In HeLa, Caco-2 and MEF cells, signaling in response to C3-coated wild-type bacteria was detected (Figure 5E). Signaling was significantly more pronounced in the ΔSifA mutant, which has reduced vesicular integrity and a greater propensity to enter the cytosol(29) (Figure 5F). Together, these data show that a variety of different pathogens can be detected by the presence of intracellular C3.

Viral antagonism to intracellular complement immunity

The hallmark of an antiviral response is that it should exert selection pressure on pathogens to evolve and escape it(30). To investigate the possibility of viral antagonism we compared the activation of immunity by complement between different viruses. We observed that whilst there was complement mediated NF-κB activation upon infection with viral particles like AdV and HPV (Figure 6A), this was significantly weaker during infection with viruses such as hAstV, PV and HRV. This suggests that these viruses have strategies that allow them to evade detection by C3. hAstV is known to inactivate complement component C1, limiting the amount of complement deposition(10). UV-inactivation of HRV restored complement mediated NF-κB activation to the levels of replication-deficient vectors, AdV and HPV, suggesting that an encoded component was antagonizing C3-mediated signaling (Figure 6A).

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.

HRV expresses a cytosolic 3C protease (HRV 3C Pro) that we hypothesized to mediate antagonism, given previous observations of 3C-mediated cleavage of RIG-I(31). Using in silico analysis of potential 3C cleavage sites(32), both HRV 3C Pro and PV 3C protease (PV 3C Pro) were predicted to cleave complement C3. To test whether expression of 3C proteases enables viruses to inhibit intracellular C3 signaling, we incubated AdV+C3fBfD with albumin or with recombinant HRV 3C Pro (Ext HRV 3C), and observed cleavage of C3 by immunoblot (Figure 6B). HRV 3C Pro did not cleave the viral proteins, but removed the larger complement-bound band (Figure 6C). Next, we expressed HRV 3C Pro or PV 3C Pro in HEK293T cells prior to infection with AdV+Serum. Expression of either 3C protease reduced NF-κB induction to levels observed with AdV+HI Serum and AdV+Serum+CVF, suggesting that complement signaling had been prevented (Figure 6D). Indeed, expression of either protease was sufficient to cause AdV to behave similarly to HRV (AdV+HRV 3C Pro, AdV+PV 3C Pro; Figure 6D). To confirm that the observed effect of 3C protease was due to cleavage of complement and not a factor inside the cell, AdV incubated with different sera was treated with recombinant 3C protease before pelleting the virus to remove the protease, before addition to HEK293T cells (Figure 6E). Pre-infection treatment with recombinant 3C proteases (Ext HRV 3C and Ext PV 3C) perturbed NF-κB induction similarly to expression inside target cells. In addition to antagonizing sensing, we observed that HRV 3C Pro or PV 3C Pro were also effective in preventing complement-mediated restriction (Figure 6F). In order to permit escape from intracellular complement immunity, 3C Protease must be synthesized and act rapidly after infection. When we blotted for C3 after infection, full cleavage was noted for HRV+C3fBfD within 45 minutes (Figure 6G) however, no changes for AdV+C3fBfD were observed after 2h. Overexpression of HRV 3C Pro was sufficient to confer C3 cleavage during AdV infection. These data suggest that cleavage of C3 is a fast and effective method of evading intracellular complement immunity.

We further hypothesized that if 3C protease allows viruses like PV or HRV to evade intracellular complement immunity then inhibition of 3C protease should make these viruses susceptible. To test this, we used rupintrivir, a specific, non-reversible inhibitor of the HRV 3C protease(33). Its addition to cells infected with HRV increased the levels of complement-mediated signaling so that HRV+Rupintrivir behaved like viruses lacking complement antagonism (Figure 6H). Rupintrivir treatment was also sufficient to make PV visible to complement sensing. When cells were treated with rupintrivir, this lead to a loss of C3 cleavage by HRV (Figure 6I). To investigate the kinetics of antagonism, rupintrivir was added at various times after infection with AdV, HRV or PV and its effect on signaling determined. The data show that rupintrivir inhibits a process that antagonizes complement-mediated sensing within the first hour of infection (Figure 6J). This timescale matches the kinetics of C3 cleavage by 3C. By 1.5h post-infection, addition of rupintrivir was unable to restore heat labile signaling, consistent with C3 cleavage being completed by this time. Rupintrivir had no impact on complement-mediated AdV sensing. These rapid kinetics are similar to those previously reported for PV 2A protease(34), formed from the same polypeptide as 3C protease. Complement also activates cell surface signaling in professional cells. Importantly, virally encoded 3C antagonism did not prevent this response. Rupintrivir had no effect on signaling in THP-1 cells infected with HRV+Serum, a finding consistent with their extracellular detection of the C3-coated virus (Figure 6K). Our data suggest that rupintrivir could be used therapeutically to enhance host detection of 3C-expressing viruses. We found that NF-κB induction by complement, facilitated by rupintrivir treatment, was sufficient to induce IFN-β secretion upon HRV infection (Figure 6L). Together, these data suggest that viral expression of 3C protease cleaves complement C3 inside the cell to disable signaling and restriction. Inhibition of 3C protease counteracts this effect.

Complement-mediated signaling is independent of known PRRs, but requires MAVS

The ability of complement C3 to enable sensing of a wide variety of different pathogens suggests that a particular PAMP is not required for detection. To test this, we blocked Toll-like receptor activity by inhibiting MyD88 and TRIF (Figure 7A), as well as knockdown of RIG-I and MDA5 (Figure 7B, S1D). In addition, we inhibited Syk (Figure 7C), which blocks signaling through Fc-receptors and type II C-type lectin receptors, and depleted cells of STING, the endoplasmic reticulum-associated adaptor involved in cytosolic DNA detection (Figure 7D, S1E). All of these perturbations to known signaling pathways had no effect on complement-mediated signaling.

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.

However, depletion of MAVS abolished the heat labile component of signaling, suggesting that MAVS is required for C3-mediated immune activation (Figure 7D, S1E). Sendai virus has been reported to activate MAVS activation by inducing aggregation(35), as detected by microscopy and by semi-denaturing gel electrophoresis. We carried out microscopy for MAVS and mitochondrial marker TOM20 in cells treated with PBS, AdV, C3fBfD or AdV+C3fBfD 6h post-infection (Figure S2A), and noted no apparent aggregation at this time point. This is consistent with published literature, in which aggregation of MAVS only occurs after 9 hours of infection(35). Overexpression of MAVS lead to increased protein levels but the same localization was observed (Figure S2B). We carried out semi-denaturing gel electrophoresis, but were not able to detect aggregation, except when MAVS was overexpressed (Figure S2C). Thus we observe rapid MAVS-dependent signaling that is independent of aggregation, with similar kinetics to that observed during MAVS-induced IRF3 dimerization(36) and IkBa phosphorylation(37).

Due to the involvement of MAVS in C3 sensing, we investigated whether TRAF proteins are required for signal transduction. Depletion of TRAF6 reduced complement-mediated NF-κB signaling (Figure 7E, S1F), although simultaneous knockdown of TRAF2, 3, 5, and 6 (siT2,3,5,6) was required to abolish signaling, similar to published data(38). Given the importance of TRAF6, we also investigated the adaptor p62(39). Depletion of p62 (Figure 7F) lead to a similar loss of NF-κB induction as that from TRAF6 knockdown. Individual depletion of TRAF proteins had a partial effect on IRF activation (Figure 7G), whilst siT2,3,5,6 depletion was completely inhibitory, demonstrating redundancy. In addition, knockdown of TBK1 or inhibition with BX795(40) prevented IRF activation (Figure 7H), demonstrating that TBK1 is important in C3-mediated signaling. Together, these data demonstrate that intracellular C3 activates MAVS with downstream signaling proceeding via the TRAF proteins in a partially redundant manner.

Intracellular antibody and complement cooperate to activate signaling

TRIM21 has previously been identified as a PRR for cytosolic antibody(1, 4) and depletion of TRIM21 (Figure S1C) resulted in decreased signaling from AdV+Serum (Figure 8A). However, TRIM21 depletion only decreased the heat stable component of signaling, because only AdV+IgG, and not AdV+C3fBfD signaling was affected. This confirms that while TRIM21 is a receptor for antibody, it is not a receptor for complement C3. Co-depletion of TRIM21 and MAVS abolished signaling from AdV+IgG, AdV+C3fBfD or AdV+Serum, suggesting that the ability of intracellular serum proteins to induce signaling (Figure 1A) is entirely dependent on these two pathways.

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.

Importantly, we noted that knockdown of MAVS did not have any effect on Adv infection in the presence of serum, whilst knockdown of TRIM21 increased infection (Figure 8B) consistent with loss of antibody dependent intracellular neutralization. However, neither MAVS nor TRIM21 depletion had any effect on the restriction of AdV+C3fBfD (Figure 8C). This suggests that C3 mediates an effector response that is independent of MAVS and TRIM21.

C3 signaling and restriction contribute independently to reduce viral infection

Next we investigated the relationship between C3 mediated signaling and restriction. Transfection of complement C3 coated beads was sufficient for NF-κB induction and this activity was dependent upon MAVS and TRAF6 (Figure 8D), demonstrating that PAMPs are not required for C3 detection. Moreover, there was strong induction of IRF3, 5, and 7 in cells where restriction was prevented using a proteasome inhibitor (Figure 8E). These data suggest that C3-mediated detection is not dependent upon viral components liberated during restriction. Finally, restriction of AdV was not affected by addition of the IκB inhibitor, panepoxydone (Figure 8F), which previously inhibited signalling (Figure 3A), suggesting that restriction is also independent of cell signaling. Thus C3 mediates independent signalling and restriction pathways, as has been shown previously for antibody(1, 2, 4).

While the above data show that C3 mediates an effector response that is independent of signalling, we investigated whether signaling leads to an antiviral state that also contributes towards reducing viral infection. Supernatant was collected from HEK293T cells 3 days after challenge with PBS, AdV, C3fBfD or Adv+C3fBfD and transferred to fresh uninfected HeLa cells. These uninfected cells were then challenged with an interferon-sensitive reporter virus (Sindbis with a GFP transgene). Supernatant from AdV+C3fBfD infected cells was sufficient to protect cells from Sindbis infection (Figure 8G), in a MAVS dependent manner. Adenovirus is relatively resistant to interferon treatment(41) but rhinovirus is interferon sensitive(42). To investigate the relative contributions of C3-mediated signaling and restriction on a spreading infection, we added epoxomicin or panepoxydone during HRV infection of HeLa cells treated with control or MAVS-targeted siRNA (Figure 8H). Epoxomicin reversed the ability of complement to restrict HRV, in line with the finding for AdV. However, panepoxydone and MAVS depletion also partially reversed restriction, suggesting that signaling in response to C3 contributes to the antiviral state. These data demonstrate that C3 elicits a direct antiviral restriction mechanism and promotes signaling which induces an antiviral state.

Discussion

In this study, we investigated whether extracellular serum proteins mediate intracellular immune responses inside non-immune cells upon pathogen infection. We found evidence for intracellular pathogen detection via the sensing of attached complement. Complement C3 is covalently deposited onto pathogens in the extracellular space. During infection, pathogens carry C3 inside the cell, activating innate immunity and mediating a restriction pathway that degrades virus. Key features of this intracellular complement immune response are that it is cell type independent and requires translocation of C3 into the cytosol. This allows pathogens to be sensed during their natural infection pathway rather than relying on the capture of immune complexes by professional cells. The importance of this system is suggested by the evolution of countermeasures that allow certain viruses to antagonize it. Both rhinovirus and poliovirus have evolved a mechanism to evade C3 detection, by cleaving C3 using an encoded 3C protease.

C3 signaling was independent of known PRRs, e.g. Toll-like receptors and RIG-like receptors and occurred for DNA and RNA non-enveloped viruses and intracellular bacteria, suggesting that exposure of PAMPs is not directly responsible. Detection was dependent on MAVS, activating transcription factors and proinflammatory cytokine secretion. The minimum requirement for NF-κB activation was found, comprising reconstituted alternate pathway components (C3, factor B, factor D) bound to beads and transfected into cells.. Importantly, C3-coated AdV only initiated signaling when intracellular and post-endosomal, suggesting that the initial receptor is cytosolic.

Sensing of complement-coated virus results in a restriction response that leads to degradation of virus and inhibition of infection. This process involves the proteasome, similar to the restriction phenotypes following recognition of retroviral capsid by TRIM5α(43), and antibody by TRIM21(1, 4). As with antibody, complement-mediated degradation also requires the AAA ATPase VCP(3).

Taken together with our previous work on intracellular antibodies(1-6), the data here suggest that serum proteins may have an important role in detecting breaches in the physiological barrier of cell membranes. C3 has recently been described as mediating immunity to Chlamydia psittaci, an obligate intracellular bacterium in mice(44). In addition, C1q has been implicated as providing an intracellular signaling role by interacting with RIG-I and MDA5(45). Whether these effects are related to the signaling described in this study is still to be investigated. Ultimately, there may be a number of host proteins and corresponding set of receptors that stimulate immunity based on a system of topological displacement.

The discovery that picornaviruses, e.g. HRV and PV encode a 3C protease capable of cleaving C3 and inactivating its intracellular function, has implications for treatment strategies. HRV is particularly sensitive to interferon(42), and treatments that prevent the virus from suppressing interferon induction could be highly effective. Rupintrivir is used to control viremia and has been reported to decrease cytokine production(46). However cytokines such as IL-6 and IL-8 were measured 3 days post infection, by which time rupintrivir may have reduced viremia, resulting in a complex phenotype. In our study, rupintrivir was removed 3 hours post-infection, resulting in the measurement of responses from a single-round of infection and unbiased by differing levels of replication. We propose that in vivo, rupintrivir may have dual functions, increasing the complement-mediated detection of virus and hence production of interferon-β as well as inhibiting the production of progeny virions by interference with polypeptide cleavage.

Finally, this system illustrates how a single DAMP can lead to the simultaneous detection and restriction of a pathogen. Signaling in response to C3-coated pathogens is independent of PAMPs, and does not require proteasome-dependent restriction to reveal molecular determinants. Restriction of the pathogen occurs immediately upon infection and is not dependent on the simultaneous activation of cell signaling. However, these two responses cooperate to enable the efficient reduction of spreading infection. While this is a relatively new concept in immunology, it is exemplified by the receptors TRIM5α(43, 47), TRIM21(1, 4) and tetherin(48, 49). These systems allow the cell to capitalize on a single sensing event to elicit multiple immune responses.

MATERIALS AND METHODS

Cells

HEK293T, HeLa, Vero, Caco-2, FEA, MDCK II and MEF cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100U/ml penicillin and 100μg/ml streptomycin. MEF cells were obtained from C57BL/6 mice as previous(5). THP-1 cells were maintained in RPMI-FCS with penicillin/streptomycin as above and 20nM 12-O-Tetradecanoylphorbol-13-acetate (TPA). Normal Human Lung Fibroblasts (NHLFs) from Lonza were maintained in Fibroblast Growth Medium 2 (Lonza), supplemented with 10% fetal calf serum, 0.1% insulin, 0.1% amphotericin-B and 0.1% gentamicin according to the manufacturer’s instructions.

Viruses and bacteria

E1, E3-deleted adenovirus vector bearing a GFP transgene (AdV) was from Viraquest Inc. Wild-type adenovirus 5 (WT AdV), human rhinovirus 14 (HRV), attenuated poliovirus 2 (PV), enterovirus 71 (EV71) were all from ATCC, grown in HeLa cells and cell-free supernatant was harvested and frozen 8 d post-infection. Human papillomavirus 16 L1/L2 virus-like particles (HPV) were produced with plasmids from M. Müller, German Cancer Research Center, following the protocol of Buck and Thompson(50). Human Astrovirus 1 molecular clone pAVIC(51) (hAstV) was provided by I. Goodfellow, University of Cambridge, and recovered following the method of Fuentes et al(52). Respiratory syncytial virus GFP-expressing molecular clone rgRSV(224)(53) (RSV) was provided by M. Peeples, Ohio State University. RSV was expanded in HeLa cells and harvested 2 d post-infection. Feline calicivirus strain F9 (FCV) was provided by D. Brown, University of Cambridge. FCV was expanded in FEA cells, harvested 24h post-infection. Coxsackie virus B3 molecular clone expressing a GFP transgene (CVB), eGFP-CVB3(54) was obtained from J.L. Whitton, Scripps Institute. Sindbis virus–GFP (Sindbis) vector was produced by in vitro transcription of linearized plasmids DH-BB and pSin-eGFP with mMessage mMachine SP6 kit (Life Technologies) and transfection of RNA into 293T human embryonic kidney cells with Lipofectamine 2000. WT AdV, HRV, CVB were prepared by CsCl banding(4), and HPV by banding in OptiPrep (Sigma). PV, EV71, hAstV, RSV, Sindbis were used directly from cell-free supernatant, with FCV by pelleting cell-free supernatant through 30% sucrose. Viruses were quantified by the TCID₅₀ method, or GFP where possible. Wild-type (strain 12023) or ΔsifA Salmonella enterica enterica serovar Typhimurium were prepared as previous(1).

Serum and serum treatments

Normal human serum (Serum), as well as the complement series depleted serum (Serum-C1, C2, C3, C5, C6) were obtained from Sigma. Rabbit serum was also obtained from Sigma. Antibody-depleted serum (Serum-Ig), with the control serum were from SCIPAC. Serum depleted of C4 (Serum-C4), factor B (Serum-fB) and factor D (Serum-fD), along with matching control serum was from Quidel. Mouse and cat serum were from Equitech Bio Inc. Heat inactivation was carried out at 52°C for 45 minutes. EGTA treatment was by adding 0.2M EGTA in 0.2M MgCl₂. Purified complement C3, factor B and factor D were from Millipore. Cobra venom factor (CVF) was purified from Naja naja kaouthia venom (MP Biomedicals) using the method of Vogel and Muller-Eberhard(55). Recombinant human rhinovirus 3C protease was from Expedeon Enzymes, and recombinant poliovirus 3C protease prepared by I. Goodfellow, University of Cambridge. Serum was used at the maximum concentration which had no impact on entry, as found by the endosomal disruption assay (typically 1/5 to 1/10 dilutions in PBS); with depleted serum verified using sheep red blood cell hemolysis assays(56).

Antibodies

Serum components binding to AdV ELISAs; goat anti-human IgM HRP, goat anti-human IgA HRP, goat anti-human IgG HRP, mouse anti-human IgD HRP, and mouse anti-human IgE HRP were obtained from Abcam. Detection of human C3b, C4b, Mannan-binding lectin, and C-reactive protein were modified from ELISA detection kits from Abcam. Pentraxin 3 detection was by modified ELISA kits from Hycult Biotech. Immunoblot of CD11b, CD11c, CD18, CD35, CD46, CD55, p62 and Complement C3 was by antibodies from Abcam. Immunoblot of RIG-I, MDA5, phosphorylated IKK, total IKK, phosphorylated IκB, total IκB, phopshorylated NF-κB p65, total p65, TRAF2, TRAF3, TRAF5, TRAF6 and GAPDH (loading control) used antibodies from Cell Signaling Technologies. Immunoblot for STING and MAVS used antibodies from Santa Cruz Biotechnology. Immunoblot and immunofluorescence for AdV hexon used polyclonal goat anti-adenovirus 5 antibody from Millipore. Immunofluoresence used MAVS and TOM20 antibodies from Santa Cruz.

Small interfering RNA knockdown

TRIM21 siRNA was carried out as previous(4). siRNA against RIG-I, MDA-5, STING, MAVS, TBK1, TRAF2, TRAF3, TRAF5, TRAF6 were from Santa Cruz Biotechnology. siRNA against CD46 and CD55 were from Origene. p62 siRNA was from Life Technologies. siRNA was transfected using RNAiMAX from Life Technologies.

Inhibitors

Pepinh-MyD and Pepinh-TRIF (Invivogen) were used alongside the supplied control peptide at 50μM. 1μM 5Z-7-Oxozaeanol (Sigma), 2μg/ml panepoxydone (Enzo Life Sciences), 200nM IKK Inhibitor VII (Millipore), 1μM Bafilomycin A1 (Santa Cruz Biotechnology), 20nM CID1067700 (Millipore), Epoxomicin (Millipore) was added at 2μM, KU55933 (Millipore) was used at 10μM, Gö6976 (Millipore) at 5μM, DBeQ (BioVision) at 8μM and rupintrivir (Santa Cruz Biotechnology) was used at 2μM, were all added 1h prior to infection.

Plasmids and reporter constructs

Luciferase reporter cell lines were produced by transfection of pGL4.32 NF-κB (Promega). NHLFs transduced with Cignal Lenti NF-κB (Qiagen). Minimal-CMV promoter driven nano-luciferase plasmid, pNL1.1CMV was from Promega. Expression constructs for HRV 3C Protease and PV 3C Protease were from Origene. Flag-MAVS was cloned into pcDNA3.1(+) using NotI and XhoI primers: forward 5′-ACGTGCGGCCGCCACCATGGACTACAAAGACGATGACGACAAGATGCCGTTTGCTGAAGACAAGAC-3′ and reverse 5′-GGCGTCTGCACTAGTGACTCGAGTCTA-3′. Plasmids were transfected using Lipofectamine 2000 (Life Technologies).

Luciferase reporter assay

Cells were plated at 1 × 10⁴ per well in 96 well plates. The day after plating, virus and serum were incubated 1:1 for 1h before addition to cells. Viruses were added at the following titres: AdV, 1.5 × 10⁶IU per well; WT AdV, 4.22 × 10⁶IU per well; HPV, L1/L2 protein at 1.92μg per well; hAstV, 7.5 × 10⁵IU per well; FCV, 2.8 × 10⁵IU per well; HRV, 3.16 × 10⁶IU per well; PV, 2.0 × 10⁶IU per well; CVB, 2.0 × 10⁶IU per well; EV71, 2.0 × 10⁵IU per well; RSV, 2.0 × 10⁵IU per well. For positive controls, 10pg/ml recombinant TNF or 50pg/ml LPS was used. Cells were incubated for 6h (18h for HPV) at 37°C, before addition of Steadylite Plus luciferase reagent (Perkin Elmer) and reading on a BMG Pherastar FS platereader. For assays involving pelleting, AdV and PBS or serum were mixed before layering onto 30% sucrose and spinning for 4h at 28,000×g at 4°C, resuspending pellet in PBS. For transfection of beads, 0.25μm biotin-coated latex beads (Sigma) were incubated with 500μg/ml C3, factor B, and factor D (Sigma) or PBS for 1 hour. Beads were transfected using Lipofectamine 2000 (Life Technologies). For infections with S. Typhimurium, overnight stationary phase cultures were diluted 1/33 into 5ml fresh LB medium and grown at 37°C, 400rpm for 3.5h. A 1/500 dilution of these cultures were incubated with 250μg/ml C3, factor B, and factor D for 15 minutes, before addition of 10μl mix onto cells. Luciferase was read 7 h post-infection.

ELISAs

Serum binding ELISAs carried out incubating AdV+Serum for 1h, before ultracentrifugation at 28,000×g for 4h at 4°C through 30% sucrose. The pellet was resuspended in PBS, before binding to polystyrene high-binding Microlon plates (Greiner) overnight, blocking for 2h with Marvel, before addition of serum. Quantification was carried out using kits listed in antibodies section, with subtraction of signal from Marvel blocked wells. NHLF cells were plated and infected in the same manner as for luciferase reporter assays. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 in the plate 4h post-infection and abundance of phosphorylated proteins was measured using the NF-κB signaling kit (Cell Signaling Technologies). For analysis of cytokine production, supernatant was harvested 24h post-infection and analyzed by ELISA kits: IL-6 (Life Technologies), TNF (Life Technologies), CCL4 (Abnova), and IFN-β (Life Technologies). Plates were read using a SpectraMAX 340PC (Molecular Devices) at 450 and 650 nm.

Endosomal Disruption Assay

Endosomal disruption assay was modified from Seth et al(57). Cells were plated at 1 × 10⁴ per well in 96 well plates, in DMEM containing 12μg/ml pNL1.1CMV (Promega). Virus mixes were added as for the NF-κB reporter. Cells were left for 18h post-infection at 37°C, before addition of NanoLuc reagent (Promega) and reading on a BMG Pherastar FS platereader.

Confocal microscopy

For intracellular C3 visualization, HeLa cells were plated on glass coverslips and permitted to adhere overnight. Purified C3 was labeled with AlexaFluor488-SDP Ester (Life Technologies) at 4°C for 2h, before storage at −80°C. AdV was incubated with 250μg/ml labelled C3, factor B and factor D for 15min, before infection for 30min. For MAVS activation visualization, HEK293T cells were transfected with pcDNA3.1-EV or pcDNA3.1-FLAG-MAVS 48h prior to infection using Fugene 6. Cells were plated on glass coverslips pretreated with CellTak (Becton Dickinson) and permitted to adhere overnight. AdV was incubated with 250μg/ml of C3, Factor B and Factor D, or AdV was incubated with PBS, or C3, factor B and factor D was incubated alone for 1h, before addition to cells for 6h. Cells were washed and fixed with 4% paraformaldehyde, before permeabilization with 0.1% Triton X-100 and blocking with 5% BSA. Immunostaining for AdV was by anti-adenovirus 5 goat polyclonal antibody (Millipore), MAVS was by mouse monoclonal (Santa Cruz) and TOM20 was by a rabbit polyclonal (Santa Cruz). Coverslips were mounted on medium containing 4′ 6-diamidino-2-phenylindole (DAPI) and imaged using a Zeiss 63X objective on a Jena LSM 710 microscope (Carl Zeiss MicroImaging).

Neutralization Assay

HeLa cells were plated at 1 × 10⁵ per well in 6 well plates. The day after plating, AdV and sera were incubated 1:1 for 1h before addition to cells. AdV was infected with 3.75 × 10⁴ IU per well. 18h post-infection, cells were harvested and GFP positive cells enumerated by flow cytometry using a BD FACS Calibur 2. Interferon treatment was with addition of 1000 U/ml Interferon-α (Sigma), 24 h prior to infection.

Fate-of-Capsid Experiment

HeLa cells were plated at 1 × 10⁵ per well in 6 well plates. The day after plating, AdV and sera were incubated 1:1 for 1 h before addition to cells. AdV was infected with 7.5 × 10⁴ IU per well. Cells were harvested by scraping at 0, 1, 2, 4 and 6 h post-infection, followed by immunoblot for AdV.

MAVS Aggregation

MAVS aggregation was carried out as previously described by Hou et al.(35). Briefly, HEK293T cells were challenged with PBS, AdV, C3fBfD or AdV+C3fBfD for 6h, after which they were harvested and mitochondrial extracts produced by douncing and ultracentrifugation. HEK293T cells expressing Flag-MAVS was harvested 3d post-transfection. Samples were controlled for total protein, and ran in Tris-Acetate gels as semi-denaturing gel electrophoresis, with immunoblot carried out for MAVS.

Transfer of Media Experiment

Sindbis experiment was carried out as previously described(1). Briefly, HeLa cells were infected with PBS, AdV, C3fBfD or AdV+C3fBfD. Cell supernatant was collected three days post-challenge, and used to treat fresh ‘reporter’ HeLa cells. Other ‘reporter’ HeLa cells were provided fresh media (DMEM), or were stimulated with 1000U/ml Interferon-α (Sigma). The following day, cells were infected with Sindbis virus at MOI=0.3, before enumeration of GFP positive cells one day post-infection.

Restricting Titre Experiment

Cells were plated at 5 × 10³ per well in 96 well plates. Serial dilutions of 250 μg/ml C3, factor B and factor D were incubated with 20×TCID₅₀ units HRV for 1h before addition to cells. DMSO, MG132 or panepoxydone were added 1h prior to infection, and cells were washed 16h post-infection into DMEM supplemented with 2% fetal calf serum. 7 days post-infection, a MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (Sigma), was performed, permeabilizing cells by DMSO, measuring absorbance with a SpectraMAX 340PC (Molecular Devices) at 540 nm.