A Cellular Model Of Infection With Influenza Virus H1N1 And Identification Of Host Factors Interacting With The Viral Nucleoprotein NP In Macrophages

Influenza A virus(IAV),the causative agent of the zoonotic respiratory infection Influenza,is a segmented single-stranded negative-strand RNA virus belonging to family.IAV is highly contagious,seriously endangering human health and causing serious public health safety.Macrophages are multifunctional immune cells that play a key role in the control of immune responses to virus infection.While being mainly involved in pathogen recognition and clearance,macrophages can also serve as the direct target cells for some viruses.These macrophages are utilized by viruses to serve as vessels for dissemination,long-term persistence within tissues and virus replication,thus facilitating the establishment and spread of viral infections.Therefore,macrophages have a potential as an important tool and as a cellular model for studying the mechanism of viral infection and host cell immune responses.However,such a cellular model for influenza virus infection and replication in macrophages is missing.To this end,we set out to establish an IAV infection model in mouse bone marrow macrophages.By using this model,we characterized the infection and replication of IAV and its mediated signaling events in macrophages.Western-blot was used to detect the expression of influenza virus proteins NP and NS1 in macrophages at protein level,and qRT-PCR was used to detect the expression of virus-specific mRNA.We show that influenza virus H1N1 can establish infection and replicate in mouse bone marrow-derived macrophages(BMMs).The replication kinetics of influenza virus in mouse BMMs was characterized over a time course of 30 hours post infection(hpi),showing the replication of influenza virus peaks at 24 hpi,and decreased thereafter.Further,by Immunofluorescence the NP antigens were detected in peritoneal macrophages and in the macrophages in lungs/spleen from IAV-infected mice.In addition,liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to identify host binding partners for IAV nucleoprotein NP,which were enriched in IAV-infected BMM by immunoprecipitation.Preliminary analysis of the protein profile identified in anti-NP immunoprecipitates by mass spectrometry suggested that some proteins of BMMs interact with NP protein are involved in signaling pathways such as RIG-I receptor and Fc? receptor.This provides a fundation for further study of the mechanisms of influenza viruses replication and the antiviral immune responses in macrophages.Overall,this study successfully established a cellular model for influenza virus H1N1 infection in macrophages.Using this model,the replication kinetics of influenza virus H1N1 in macrophages,and the impact of LPS/TLR4 signaling pathway on IAV replication in BMMs were characterized.In addition,the potential binding partners of influenza virus nucleoprotein NP in macrophages were initially identified.This work may provide insights into the cellular mechanisms of the influenza virus replication and immune escape,and host anti-influenza virusimmune responses.