Influenza A Virus (IAV) subtypes, A/England/195/2009 (pH1N1) and the A/HK/1174/99 (H3N2), were obtained as described recently (32, 33). H1N1 and H3N2 pseudotyped viral particles were produced using the plasmids obtained from Addgene (32, 33). pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1-flag (N1 from swine H1N1 A/California/04/09), and pcDNA H3 (from A/Denmark/70/03 (H3N2) (Invitrogen) were purchased commercially. Plasmid pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)] was provided by Dr Nigel Temperton (University of Kent). Anti-influenza viral antibodies and recombinant viral proteins were obtained from BEI Resources, National Institutes of Health, USA (32, 33).

The IAV subtypes investigated in this study were produced as previously described (32). Briefly, Madin-Darby Canine Kidney Cells (MDCK) were cultured in growth medium (DMEM-F/12 with GlutaMAX™ Supplement (Gibco) + 10% v/v Heat Inactivated Fetal Bovine Serum (FBS) (Gibco) + 1% v/v Penicillin-Streptomycin (PS) (Gibco) to 70% confluence in a 175 cm² flask (Fisher Scientific). The cells were washed with Phosphate-buffered saline (PBS) (0.01M sodium phosphate, 0.0027M KCl, and 0.137M NaCl, pH 7.4 at 25°C) (Fisher Bioreagents) to remove any residual growth media. The cells were then challenged with H1N1 and H3N2 IAV subtypes diluted in serum-free DMEM (Gibco) for 1h at 37°C. Unbound virions were washed away with PBS and the cells were then incubated for 3 days at 37°C in infection medium [DMEM + 1% penicillin/streptomycin + 0.3% w/v bovine serum albumin (BSA) + 1 µg/ml of l-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-Trypsin] (Sigma-Aldrich). Post incubation, the viral particles were harvested from the supernatant after centrifugation at 12,000 × g at 4°C for 45 min. The titre of the virus isolated was determined to be 1 × 10⁶ PFU/ml based on Tissue culture Infective Dose (TCID₅₀) Assay using the Reed–Muench method (31).

Pseudotyped lentiviral particles used were produced as previously described and characterised by western blotting (32). Briefly, HEK 293T cells were cultured in growth media to 70% confluence at 37°C under 5% v/v CO₂. Cells were co-transfected using 20 µg of plasmids described in Table 1 . The media was centrifuged at 5,000 × g for 10 min at 4°C to remove cell debris and the pseudotyped particles were harvested. The pseudotyped particles were concentrated by ultracentrifugation at 25,000 × g for 3 h at 4°C (33) and stored at -80°C until further use.

Human native properdin was purified as previously described (22, 34). Briefly, IgG-depleted human serum (using Protein G-Agarose) was passed through a column containing CNBr-activated Sepharose (GE Healthcare, UK), coupled to a monoclonal antibody against human properdin (specific to TSR3). The column was washed with 3-bed volumes of HEPES buffer (10 mM HEPES, 140 mM NaCl, 0.5 mM EDTA, and pH 7.4), and the matrix-bound properdin was eluted using 3 M MgCl₂. The peak fractions were dialysed against HEPES buffer overnight at 4°C. Minor contaminants were removed by passing through the HiTrap Q FF-Sepharose ion exchange column (GE Healthcare, UK).

Human peripheral blood neutrophils were prepared by dextran sedimentation using heparinised whole blood obtained from healthy donors, in accordance with King Faisal Specialist Hospital and Research Centre Research Advisory Council (IRP) regulations using Ficoll-Paque (35). Contaminating RBCs were removed by hypotonic lysis with isotonic NH₄Cl. The remaining cells were suspended in Krebs-HEPES medium (pH 7.4) containing 120mM NaCl, 1mM CaCl₂, 1.2mM MgSO₄, 4.8mM KCl, 1.2mM KH₂PO₄, 25mM HEPES, and 0.1% BSA. The neutrophils were purified through neutrophil isolation medium (Cardinal Associates, Santa Fe, USA) to yield 98% purity and 99% viability, as measured by flow cytometry and trypan blue exclusion tests. Isolated cells were suspended in Krebs/HEPES buffer at 5 × 10⁶ cells/ml and 0.1% BSA, pH 7.4.

Neutrophils were stimulated with H1N1 and incubated at 37°C overnight with gentle shaking. IL-6 (10 ng/ml) and the chemotactic peptide N-Formylmethionine-leucyl-phenylalanine (fMLP) (1µM) were used as positive controls. The neutrophils were centrifuged for 5 min at 10,000 x g, and supernatant was collected and concentrated using 10KD cutoff Amicon^® ultra-4 centrifugal filter units (UFC805024, Millipore). Samples were spiked with a constant amount of actin (10µg/ml), which were used as gel loading controls. Secreted properdin was detected by Western Blotting using rabbit anti-human properdin (1.19 mg/ml) (14); actin was detected by anti-actin antibody (mAb 4970, Cell Signaling Technology, MA, United States). ECL was performed using standard Horseradish peroxidase (HRP) labelled secondary antibodies.

ELISA and far-western blotting analysis were performed to determine the direct interaction of IAV subtypes with human properdin (32). Briefly, ELISA was conducted by coating constant concentration (20 µl) of whole viral particles (H1N1/H3N2; 1.36 × 10⁶ pfu/ml), constant concentration (2 μg/well) of recombinant HA or NA or decreasing concentration of human properdin (5, 2.5 or 1.25 µg/well) in a 96-well plate using Carbonate-Bicarbonate (CBC) buffer, pH 9.6 at 4°C overnight. After washing out the excess CBC buffer with PBS, three times, 20 µl of concentrated H1N1 or H3N2 (1.36 × 10⁶ pfu/ml) or decreasing concentration of properdin (5, 2.5 and 1.25 µg/ml) was added to corresponding wells, followed by incubation at 37°C for 2h. The binding was detected using respective primary antibodies (1:5000); monoclonal anti-influenza virus H1 HA, A/California/04/2009 (H1N1) pdm09, Clone 5C12 (produced in vitro) (BEI-Resources, NIAID, NIH, USA), monoclonal anti-influenza A virus neuraminidase, Clone NA2-1C1 (produced in vitro, BEI-Resources, NIAID, NIH,USA, NR-50239) polyclonal anti-influenza virus H3 HA, A/Hong Kong/1/1968 (H3N2) [(antiserum, Goat) (NR-3118 BEI-Resources, NIAID, NIH, USA)], polyclonal anti-VSV-G (1 mg/ml of purified IgG; Imperial College London), or rabbit anti-human properdin (MRC immunochemistry unit, Oxford). After 3 washes with PBS-Tween 20 (0.05%) (PBST), the binding was detected using respective secondary antibodies (1:5000); goat anti-mouse IgG HRP or goat-anti-rabbit IgG HRP (Promega). The colour was developed by adding 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (50 µl/well). The reaction was terminated using 1M H₂SO₄ (50 µl/well), and absorbance was read at 450 nm using a Biorad ELISA plate reader.

For far-western blotting, a 12% v/v SDS PAGE gel with whole virus lysates and recombinant viral envelope proteins were transferred to a PVDF membrane, followed by incubation with properdin (20 μg/ml) for 1h at 4°C, and 1h at room temperature. After three washes (10 mins each) with PBST, the membrane was probed with rabbit anti-human properdin antibody (1:1000), followed by detection with goat-anti-rabbit IgG HRP (1:1000; Promega). The blot was developed using SIGMAFAST™ 3,3′-Diaminobenzidine (DAB) (Sigma) as per the manufacturer’s instructions.

A549 cells (0.5 × 10⁶) were seeded overnight in growth media. The next day, IAV particles (MOI 1) were pre-incubated with properdin (20 μg/ml) for 2h at room temperature and added to serum starved A549 cells. Post incubation at 2h and 6h, the cells were washed with PBS gently and pelleted. GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) along with DNase I (Sigma) treatment was used to extract total RNA without any DNA contaminants. The extracted mRNA was converted to cDNA using TaqMan™ High Capacity RNA to cDNA Kit (Applied Biosystems™) and stored at −80°C until needed. qRT-PCR was performed using a StepOnePlus System (Applied Biosystems™) at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The relative expression (RQ) was calculated using A549 cells infected with respective viruses as the calibrator. The RQ value was calculated using the formula: RQ = 2^−ΔΔCt. Primers for each target gene were generated for specificity using the Primer-BLAST software (Basic Local Alignment Search Tool) (36). qRT-PCR was performed using primers listed in Table 2 ; human 18S rRNA was used as an endogenous control to normalise gene expression.

Matched H1N1 or unmatched H3N2 pseudotyped particles were pre-incubated with properdin (20 μg/ml) for 2 h at RT. Serum starved MDCK cells (1 × 10⁵ cells/well) were transduced with properdin-preincubated H1N1 or H3N2 pseudotyped particles for 24h in infection medium. IAV pseudotyped particles without properdin preincubation were used as controls. Post incubation, the unbound pseudoparticles were removed, and complete media was added to the cells, followed by incubation for a further 72h for luciferase expression. Luciferase reporter activity was measured using Promega™ Bright-Glo™ Luciferase Assay System (Promega) and Luciferase Plate reader (Promega).

For measuring NF-κB activity, A549 cells (1 × 10⁴ cells/well) were seeded in 12-well plate and transfected with pNF-κB-LUC plasmid using BioT transfection kit (Bioland Scientific) as per the manufacturer’s instructions for 48h. Viral particles (MOI 1), pre-incubated with properdin (20 μg/ml) for 2h, were used to challenge the transfected A549 cells in infection media for 6h. Virus particles that were not treated with properdin were used as control. A549 cells treated with recombinant TNF-α (10 ng/ml) and IL-1β (10 ng/ml) (R&D Systems) was used as a positive control for NF-κB activation. Luciferase reporter activity was measured as described above.

Cell binding assay was performed to evaluate if the binding of the IAV virions with properdin affected the ability of the IAV virions to bind to the cell surface. Briefly, A549 cells (1 × 10⁵ cells/well) were challenged with H1N1 or H3N2 (1.36 × 10⁶ pfu/ml) viruses, pre-incubated with or without properdin (20 µg/ml) and incubated at 37°C for 2h. Following PBS washes, the cells were fixed with 1% v/v paraformaldehyde (Fisher Scientific) for 1 min at room temperature, followed by blocking with 2% w/v BSA for 2 h at 37°C. The binding was probed with either polyclonal anti-influenza virus H3 (1:5,000; BEI-Resources), or monoclonal anti-influenza H1 (1:5,000; BEI-Resources). The colour was developed by adding TMB substrate (50 µl/well) and the reaction was stopped using 1M H₂SO₄ (50 µl/well). The absorbance of the plate was read at 450 nm using an ELISA plate reader (BioRad).

Crystal structures of HA of H1N1 (PDB ID: 4M4Y) and H3N2 (PDB ID: 4FNK) in trimeric form, NA of H1N1 (PDB ID: 6Q23) and H3N2 (PDB ID: 4GZX) in tetrameric form, and human properdin (PDB ID: 6S0A) were retrieved from RCSB PDB database. The 3D structures of HA lacked the transmembrane region, and that of NA lacked the stalk and transmembrane region. The cleavage site of HA is known to be essential for adopting the active conformation (37). These residues were found to be missing in 4M4Y and 4FNK crystal structures. The missing cleavage site residues (ILE326, GLN327, SER328, ARG329 of 4M4Y and GLN327, THR328, ARG329 of 4FNK) were modelled de novo using a random tweak algorithm (38).

The above structures were cleaned to remove water molecules and other artefacts, protonated, minimised and typed with CHARMm forcefield (39).

HA and NA structures of H1N1 and H3N2 virus were docked with human properdin using the ZDOCK algorithm (40) available in BIOVIA Discovery studio release 2021 (41). 1000 top poses were selected and clustered using RMSD and interface cutoff of 10 Å. The clusters were sorted according to the cluster size and binding energy (Z-Rank score). The lowest energy poses among the top 10 clusters were selected and analysed for the intermolecular interactions.

The graphs were generated and statistical significance calculated using the GraphPad Prism 9.0 software. The statistical significance was considered as indicated in the figure legends between treated and untreated conditions. Error bars show SD or SEM as stated in the figure legends.

The release of properdin by the H1N1-challenged neutrophils were assessed by western blotting ( Figure 1 ). A clear difference in properdin levels was observed in IAV H1N1-challenged neutrophils compared to supernatant from the control sample (neutrophils alone). Neutrophils treated with IL-6 or fMLP, two well known inducers of neutrophils, were used as positive controls ( Figure 1 ). The amount of properdin produced by H1N1 challenged neutrophils compared well with its positive control, suggesting that the virus was a potent inducer of properdin released from neutrophil granules.

The ability of immobilised native properdin to bind H1N1 and H3N2 viral particles ( Figure 2A ) and vice versa ( Figure 2B ) was assessed using a direct ELISA ( Figure 2 ). Solid-phase properdin bound both IAV subtypes in a dose-dependent manner when probed with either polyclonal anti-influenza virus H3, or monoclonal anti-influenza H1. The immobilised viral particles also bound properdin in a dose-dependent manner when probed with rabbit anti-human properdin but with lower efficiency. Vesicular stomatitis virus G (VSV-G) protein pseudotyped lentivirus particles were used as a negative control RNA virus, which showed no significant binding with properdin at various concentrations tested.

Far-western blotting analysis was performed to determine properdin (20 µg/ml) binding to IAV glycoproteins ( Figure 3 ). Properdin was found to bind surface glycoproteins, hemagglutinin (HA; ~70 kDa) and neuraminidase (NA; ~55 kDa), in addition to matrix protein 1 (M1; ~25 kDa) ( Figure 3 ). Additionally, an ELISA was performed to quantitatively validate properdin binding to a constant concentration of purified recombinant H1N1 HA (2 µg/well) and H1N1 NA (2 µg/well) ( Figure 4 ). Properdin bound HA and NA in a concentration-dependent manner; properdin bound purified HA better than NA ( Figure 4 ). Again, no significant binding was observed for VSV-G pseudotyped lentivirus particles.

HA, NA and M1 are crucial proteins of IAV, which have many essential functions, including viral entry, budding of virions, IAV infectivity and replication. Since human properdin bound to HA, NA, and M1 of IAV, an infection assay was performed to assess the ability of IAV subtypes to infect A549 lung epithelial cells in the presence of properdin (20 µg/ml) ( Figure 5 ). A549 cells challenged with properdin pre-treated IAV subtypes showed differential expression of M1 mRNA transcript levels. In the case of H1N1, properdin pre-treatment down-regulated M1 expression by -0.5 log₁₀ at 6h compared to untreated cells (Cells + H1N1), suggesting that the properdin interaction with H1N1-viral particles reduced the viral replication efficacy at an early stage of the IAV-replicative cycle. However, H3N2-infected A549 cells, pre-treated with properdin, showed an up-regulation (1.7 log₁₀) of M1 expression, suggesting an enhanced replication ( Figure 5 ).

The mRNA levels of pro-inflammatory cytokines and chemokines in IAV-infected A549 cells with and without properdin (20 µg/ml) treatment were determined by qRT-PCR at 2h and 6h time points ( Figure 6A ). The relative mRNA expression levels of IL-12, IL-6, TNF-α, and RANTES were down-regulated following properdin treatment at 6h in H1N1-infected A549 cells. However, in the case of H3N2-infected cells, properdin treatment caused up-regulation of mRNA levels of IL-12, IL-6, TNF-α and RANTES, suggesting that properdin affected these cells in an IAV-subtype dependent manner ( Figure 6A ). During IAV infection, IFN-α is also one of the critical type I interferon that possesses strong antiviral and immune-modulatory properties (42). H1N1-infected A549 cells pre-treated with properdin showed a reduction in mRNA levels of IFN-α, while upregulation was observed in the H3N2-infected cells. This suggests the ability of properdin to reduce the efficacy and rate of viral replication, thus, diminishing the levels of IFN. Furthermore, nuclear factor kappa B (NF-κB), a nuclear transcription factor, is activated by IAV infection, thereby causing the overexpression of IAV proteins, including HA, NA and M1, during viral infections (43–45). In this context, the NF-κB mRNA expression was also measured in IAV-infected A549 cells following properdin treatment ( Figure 6A ). Properdin treatment downregulated NF-κB levels (-0.5 log₁₀) in H1N1-infected cells at 6h while promoting the expression in H3N2-infected cells by 2.3 log₁₀. This NF-κB suppression can be attributed to either a reduced viral titre in the case of H1N1-infected A549 cells or the possibility of IAV-NS1 acting as a suppressor of NF-κB activation (46). Furthermore, luciferase reporter assay was also performed to determine the NF-κB activation in IAV-infected A549 cells treated with and without properdin levels ( Figure 6B ). The data was consistent with the qRT-PCR analysis; a reduced NF-κB luciferase reporter activity (-11%) was observed in A549 cells infected with H1N1 pre-incubated with properdin, while enhanced activity (approximately ~3-fold) was detected in the case of H3N2 ( Figure 6B ). A549 cells treated with recombinant TNF-α and IL-1β were used as a positive control ( Figure 6C ), which showed a significant increase in NF-κB levels.

Lentiviral pseudotypes were generated to study the effect of properdin on the viral cell entry independent of other IAV components of H1N1 and H3N2 subtypes ( Figure 7 ). Approximately 73% reduction in luciferase reporter activity was observed in MDCK cells infected with H1N1-lentiviral pseudotypes pre-incubated with properdin (20 µg/ml), compared to untreated control (Cells + H1N1 lentiviral pseudotypes) ( Figure 7 ). However, an opposite effect was observed in the case of MDCK cells challenged with H3N2-lentiviral pseudotypes pre-incubated with properdin; ~2.5 -fold increase in luciferase reporter activity was seen. Thus, properdin acts as an entry inhibitor against the H1N1 subtype while it promotes entry of the H3N2.

Reduced luciferase activity in the H1N1 subtype treated with properdin (20 µg/ml) suggested that properdin might block IAV infection at the early stage of the IAV cycle. A cell-binding assay was performed to assess if properdin pre-treatment inhibited viral infection by preventing the binding of IAV virions to the A549 cell surface ( Figure 8 ). In this assay, ~40% reduction in cell binding was observed in properdin pre-treated and H1N1 challenged A549 cells. In comparison, ~20% increase in cell binding was noted with A549 cells challenged with H3N2 pre-incubated with properdin compared to cells infected with virus alone. Reduction in cell binding following properdin treatment in the case of H1N1-challenged A549 cells highlighted that properdin prevented the viral particles from binding to their cell surface receptors and thus, acting as an entry inhibitor against the H1N1 subtype.

In-silico docking analysis was performed to compare the binding affinity of properdin to HA and NA of H1N1 and H3N2. The analysis indicated that properdin could interact with HA and NA of both the subtypes. Properdin was found to exhibit a higher binding affinity towards HA and NA of H1N1 than H3N2, as inferred from the binding energy scores ( Table 3 ). Analysis of the top docked poses revealed that properdin engaged in the hydrogen bond interactions with three residues of the cleavage site - SER325, ILE326, GLN327 present in HA of H1N1, while a weak hydrophobic interaction involved with LYS326 present at the cleavage site of HA of H3N2 in the top docked pose ( Table 3 ; Figure 9A ). Properdin was also found to interact with the residues (LYS222, ASP225, GLU227) proximal to the sialic acid receptor binding site (GLN226, THR136, ALA137, HIS183, ASP190) (47) in HA of H1N1. These interactions were absent in the top docked pose of properdin with HA of H3N2 ( Figure 9B ).

The top docked poses of properdin with HA of H1N1 and H3N2 revealed proximal binding sites. However, this was not the case for properdin docked with NA of H1N1 and H3N2. This may be due to the lack of availability of complete crystal structures of NA of H1N1 and H3N2. In addition, differences in glycosylation patterns were evident in the NA crystal structures of H1N1 and H3N2. In H3N2 NA, the glycosylation sites were present near the sialic acid-binding sites, unlike in H1N1 NA, where the glycosylation sites were present near the stalk region ( Figure 10 ). The different overexpression systems used (Spodoptera frugiperda for NA of H1N1 and Trichoplusia ni for NA of H3N2) for elucidating the crystal structures of H1N1 and H3N2 NA could have led to the glycosylation differences observed in these structures. In the absence of the complete crystal structures of NA of H1N1 and H3N2 and the distinctly dissimilar glycosylation patterns observed in these two structures, it is difficult to be certain about inference based on the docking experiments of properdin and NA of H1N1 and H3N2.