Influenza a virus interaction with complement factor H

Abstract

Influenza A virus (IAV) is a major causative agent of respiratory tract infection in humans. Human IAVs cause seasonal epidemics, whilst avian IAV strains sporadically cross the host range barrier leading to zoonotic infections. The complement system is a component of the innate immune system that acts to remove pathogens including IAVs. Many pathogens have been reported to actively target components of the complement system to ensure their survival within the host. Therefore, this thesis aims to understand if there is an interaction between IAV and factor H (FH), a critical inhibitor of the complement system and what effect any interaction may have on IAV replication. Direct interaction between purified human FH protein and purified human H1N1 and H3N2 IAVs and avian H5N3 and H9N2 IAVs, was assessed. An interaction does occur between FH and all the IAV strains tested, and this interaction is mediated by the viral HA and NA surface protein, but not the viral M1 protein. Several technical approaches were performed to confirm that the HA protein mediates IAV binding to FH. Deglycosylation of purified IAV and FH revealed that the interaction of IAV with FH is a protein-to-protein interaction and mediated by protein to carbohydrates. Moreover, removal of glycans increased the ability of HA protein to bind to FH. IAV-FH interaction site mapping was undertaken using different fragments of recombinant FH protein and a panel of biotinylated HA peptides from of H1N1 and H3N2 IAVs. IAV binding to FH occurs in two regions on the surface of the FH protein: domains 5-7 and 15-20. Molecular characterizations of HA binding sites on the surface of the H1 and H3 HA proteins that are involved in the interaction with FH revealed that FH binds to 130-loop and the surrounding area, as well as with residues between 190-helix and 220-loop in RBS pocket of the HAs. Moreover, FH also interacts with several residues involved in fusion peptide pocket in HA1 or HA2 in both H1 and H3 HA protein. Interestingly the interaction has divergent effects on the replication of the IAV strains, marginally enhancing human H1N1 replication and dramatically restricting H3N2 replication in human A549 and THP-1 cells whilst having no effect on the replication of avian H5N3 or H9N2 viruses. The role of FH at the entry stage of IAV infection in A549 cells was analysed using a variety of different methods and this revealed that FH slowed down but did not completely inhibit H1N1 virus entry during the early stage of the replication cycle, while it completely prevented the entry of H3N2 into A549 target cells. Overall, these findings indicate that different subtypes of human and avian IAVs have binding ability to human FH that reveals a different effect on IAV replication for various strains.