Interactions of SEPT5 with host neuronal and H5N1 viral proteins during infection

Widespread infections of highly pathogenic H5N1 influenza A viruses in poultry are mainly associated with host’s immune system and viral virulence. In poultry, general systemic infections involving all organs and encephalitis are the common cause of the very high mortalities. For many viruses, the genotype and phenotype of the viruses associated with viral entry and exit play critical roles in virulence. Since cellular plasma membranes are involved in viral entry and exit, more studies need to be conducted to determine the role of cellular plasma membrane components involved especially during virus exit. The identification and detection of high level of SEPT5 protein; a cytoskeletal component associated with cellular membranes and its mRNA in H5N1-infected chicken brains and neuronal cells respectively, provided evidence for the potential importance of this protein in the pathogenesis of neurovirulence caused by H5N1. The involvement of septins in viral disease pathogenesis, however, has yet to be documented. In this study, we therefore investigated SEPT5 and H5N1 viral proteins interactions relating to viral virulence. Two distinct studies involving cellular transcripts and proteomic analyses of chicken brain tissues found that SEPT5 mRNA were up-regulated, in response to neurovirulent H5N1 avian influenza virus infection. In this study, the presence of SEPT5 proteins was detected in human neuronal cells and similarly in chicken brain tissues models where SEPT5 was shown to be present in the substantia nigra region of chicken brain. Recombinant (r)SEPT5 proteins were produced in the E.coli expression system and used in co-immunoprecipitation assay where rSEPT5 was shown to interact with other septin protein species; SEPT2, SEPT6, SEPT7 and SEPT11 found in H5N1-infected chicken brain. A novel interaction between hemagglutinin (HA1) and neuraminidase (NA) viral proteins with rSEPT5 were also identified using mammalian-2-hybrid system. These interactions were further validated with co-immunoprecipitation assays and co-localization assays confirming, that HA1 and NA proteins co-localized with SEPT5 protein in the neuroblastoma cells. The binding site of truncated-constructs of the SEPT5 gene indicated the importance of coiled-coil region on SEPT5 in facilitating binding with the viral proteins. Silencing of endogenous SEPT5 with siRNA in neuroblastoma cells confirmed its role in viral exit as reduced amount of viral mRNA was detected in the extracellular medium of SEPT5 knockdown neuroblastoma cells. Disruption of the SEPT5 protein by silencing reduced viral exit, thus reducing viral load for infection and indirectly, the virulence of the virus. Viral load is one of the factors that are associated with virulence of the virus. This is the first study conducted that showed chicken SEPT5 interacted with the HA1 and NA proteins of a highly pathogenic H5N1 influenza virus and that SEPT5 may play a role in the exit of viral particles and virulence.