C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus

Abstract

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.

Keywords: C4BP; complement; inflammation; influenza A virus; pseudo-typed lentiviral particles.

Figure 1

Characterization of purified C4BP. (A) SDS-PAGE (12% w/v acrylamide/bisacrylamide) was loaded with 5 µg of purified C4BP in lane 2, along with a protein ladder with a range of 250 to 10 kDa (Thermofisher) in lane 1. The denatured and reduced samples were run for 120 min at 90 V and stained with Coomassie brilliant blue to reveal protein bands corresponding to the α-chain (~70kDa) and a faint band corresponding to β-chain of C4BP (~45 kDa). (B) Immunoreactivity of the purified C4BP was analyzed by western blotting. A PVDF membrane with 1.25, 2.5, and 5 µg of purified C4BP in lanes 2, 3, and 4, respectively, along with a protein ladder with a range of 250 to 10 kDa (Thermofisher) in lane 1 was probed using rabbit-anti-human C4BP polyclonal antibodies (1:1,000) at room temperature for 1 h, followed by incubation with secondary goat anti-rabbit IgG HRP-conjugate (1:1,000) for 1 h at room temperature. Bands corresponding to α-chain (~70 kDa) and the β-chain (~45 kDa) of the C4BP were observed after developing the color using 3,3′-diaminobenzidine (DAB) substrate.

Figure 2

Binding interaction of immobilized C4BP to IAV subtypes (A), and immobilized IAV subtypes to C4BP (B). Microtiter wells were coated with a varied concentration of C4BP (5, 2.5, and 1.25 µg/ml) protein and incubated with constant volume of viruses (1,000 PFU/ml). Bound C4BP-IAV was probed with monoclonal anti-influenza virus H1 and polyclonal anti-influenza virus H3 antibody (1:5,000). Another ELISA was performed by coating a constant concentration of IAV subtypes (1,000 PFU/ml) and incubated with a varied concentration of C4BP protein (5, 2.5, and 1.25 µg/ml). IAV-C4BP interaction was detected using polyclonal rabbit anti-human C4BP antibodies and anti-rabbit IgG-HRP-conjugated antibody (1:5,000). A validation ELISA to show the detection of H1N1 (C) and H3N2 (D) binding to a recombinant C4BP (rC4BP) and plasma purified C4BP. Microtiter wells were coated with immobilized rC4BP and the plasma purified C4BP (5 µg/ml), and incubated with H1N1 or H3N2 (1,000 PFU/ml). The binding between immobilized proteins and IAV subtypes were detected using monoclonal anti-influenza virus H1 and polyclonal anti-influenza virus H3 antibody (1:5,000). VSV-G pseudo-typed lentiviral particles were used as a negative control; no significant binding was observed. The data were expressed as the mean of three independent experiments done in triplicates ± SEM.

Figure 3

Far Western blot analysis showing C4BP binding to H1N1 and H3N2 proteins. PVDF membrane containing H1N1 and H3N2 virus proteins, separated by SDS-PAGE was blocked with 5% w/v skimmed milk and incubated with 5 µg/ml of C4BP (A) or without C4BP (F) followed by washing steps. The membrane was probed with rabbit-anti-human C4BP polyclonal antibodies (1:1,000) at room temperature for 1 h, followed by washing and incubation with secondary goat anti-rabbit IgG HRP-conjugate (1:1,000) for 1 h at room temperature. Bands corresponding to Hemagglutinin (HA) (~70 kDa), Neuraminidase (NA) (~55 kDa), and Matrix protein 1 (M1) (25 kDa) in the case of both H1N1 and H3N2 subtypes were revealed after developing colour using 3,3′-diaminobenzidine. The identities of C4BP bound IAV glycoproteins were confirmed using separate blots that were directly probed with anti-HA (B), anti-NA (C), anti-M1 (D), or anti-NS1 (E) antibodies.

Figure 4

C4BP interacts with IAV through multiple CCP domains. ELISA showing binding of C4BP CCP deletion mutants to (A) H1N1, (B) H3N2 and (C) VSV-G: microtiter wells were coated with 5 µg/ml of the mutant C4BP protein (as described in Table 1 ). 1000 PFU/ml of H1N1 or H3N2 virus was added to all the wells. After incubation and washing steps, the bound virus was measured with either monoclonal anti-influenza virus H1, polyclonal anti-influenza virus H3 or polyclonal anti-VSV-G antibody. Results are normalised against full length C4BP α chain. The data were expressed as the mean of three independent experiments done in triplicates ± SEM. Significance was determined using the two-way ANOVA test (*p < 0.05, **p < 0.01, and ****p < 0.0001) (n = 3).

Figure 5

C4BP restricts replication of H1N1 and promotes replication of H3N2 in target human A549 cells. M1 expression of both H1N1 and H3N2 IAV after infection of A549 cells for 6 h was measured by qRT-PCR. A549 cells were challenged either with H1N1 or H3N2 (MOI 1) that were incubated with varying concentration of C4BP (10, 20, and 40 µg/ml). Cell pellets were harvested at 6 h to analyze the M1 expression of IAV. Cells were lysed, and RNA extracted was converted into cDNA. Infection was measured via qRT-PCR using M1 primers; 18S rRNA was used as an endogenous control. The relative expression (RQ) was calculated by using cells infected with respective viruses that were not treated with C4BP as the calibrator. The RQ value was calculated using the formula: RQ = 2^−ΔΔCt. Assays were conducted in triplicates, and error bars represent ± SEM. Significance was determined using the two-way ANOVA test (****p < 0.0001) (n = 3).

Figure 6

C4BP modulates viral entry in a strain-dependent manner. Luciferase reporter activity of C4BP treated MDCK cells infected with matched H1+N1 and unmatched H3+N2 lentiviral pseudo-particles. H1N1 particles exhibited a 55% decrease in viral entry compared to controls, while a 17% increase in viral entry in case of H3N2 was observed. Results are normalized against cells infected with the particles not treated with C4BP. Significance was determined using the unpaired one-way ANOVA test (****p < 0.0001) (n = 3).

Figure 7

Cytokine modulation by C4BP of IAV infected A549 cells. H1N1 or H3N2 incubated with 10µg/ml of C4BP was used to infect A549 cells. Cell pellets were harvested at 2 h and 6 h to analyse the expression of cytokines and chemokines. Cells were lysed, and purified RNA was converted into cDNA. The expression levels of cytokines IFN-α (A), IL-12 (B), NF-κB (C), TNF-α (D), IL-6 (F) and chemokine RANTES (E) were measured using qRT-PCR, and the data were normalised via 18S rRNA expression as a control. The relative expression (RQ) was calculated by using cells infected with respective viruses not treated with C4BP as the calibrator. The RQ value was calculated using the formula: RQ = 2^−ΔΔCt. Assays were conducted in triplicates, and error bars represent ± SEM. Significance was determined using the two-way ANOVA test (**p < 0.01, and ****p < 0.0001) (n = 3).