Complement-Independent Modulation of Influenza A Virus Infection by Factor H

Abstract

The complement system is an ancient innate immune defense mechanism that can recognize molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesized factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV entry, as evident from down-regulation of matrix protein 1 (M1) in H1N1 subtype-infected cells and up-regulation of M1 expression in H3N2-infected A549 cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ∼70 kDa), neuraminidase (NA, ∼60 kDa), and M1 (∼25 kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, and RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. A recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%), and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory responses, independent of its complement-related functions.

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

“…Recently, factor H, the principal regulator of the alternative pathway, and vaccinia virus complement control protein (VCP), a homologue of factor H, were found to interact with IAV in a manner similar to C4BP ( 32 ). MDCK cells infected with H1+N1pseudo-typed lentiviral particles resulted in approximately 25% and 45% reduction in luciferase activity after treatment with factor H or VCP, respectively.…”

“…Thus, using lentiviral vectors, pseudo-typed for H1+N1 and H3+N2, Murugaiah et al. demonstrated the ability of Factor H and VCP to modulate IAV entry, in a subtype dependent manner, similar to C4BP ( 32 ). As seen in the case of C4BP, Murugaiah et al.…”

“…Transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, and IFN-α were upregulated, while RANTES was downregulated following factor H treatment for the H1N1 subtype at 6 h post-infection. In the case of the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced ( 32 ). The findings of this study highlight a possible common mechanism that is used by C4BP, VCP, and Factor H that contain CCP domains in modulating the entry of IAV subtypes.…”

“…Pseudo-typed Lentiviral Particles were produced as previously described by Murugaiah et al., 2020 ( 32 ). Briefly, HEK 293T cells were cultured in RPMI 1640 (Gibco), supplemented with 10% v/v heat inactivated Fetal Bovine Serum (FBS) (Gibco), 1% v/v Penicillin-Streptomycin (Gibco) at 37°C under 5% v/v CO 2 .…”

“…The lentiviral particles in the supernatant were then concentrated by ultracentrifugation at 25,000 × g for 3 h at 4°C ( 33 ). The pseudo-typed lentiviral particles produced were characterized by western blotting ( 32 ).…”