Influenza A viruses (IAV) are prominent human pathogens and understanding the molecular biology of their replication is of utmost importance for the development novel anti-viral treatments and surveillance practices. IAV genome replication proceeds in the nucleus of infecting cells; where the virus takes advantage of host cell transcription machinery, particularly mRNA processing enzymes. The role of nuclear localization in the viral replication cycle is not clear and the viral and host cell determinants that are required for efficient nuclear import and export are an active area of study in our laboratory and others. Specifically, we are studying the role of IAV Nuclear Export Protein (NEP) in nuclear export in virus replication, biogenesis, and pathogenesis. A previously identified host nuclear export protein, CRM-1, has been identified as a critical mediator of the nuclear egress of newly replicated viral ribonucleoproteins (vRNPs). To define the virus-host interactions required for pathogenesis in humans and ferrets, the preferred animal model for IAV infection, we have cloned and expressed human and ferret CRM-1 and fused them to GFP and RFP to allow in vitro analysis of NEP/CRM-1 interactions in the context of IAV replication and biogenesis. We have developed a mammalian two-hybrid system for co-transfection of human or ferret CRM-1 and NEP from a panel of human and highly pathogenic avian influenza virus strains. This mammalian two-hybrid system has allowed for the identification of critical amino acid residues required for efficient nuclear export and virus biogenesis in human or ferret cells with a focus on the role of these later steps in replication in the dissemination of virus in the infected host or in the transmission to other susceptible hosts. In addition, using microRNA (miRNA) targeting sequences of CRM-1 and/or NEP inhibiting their expression, we examined the interaction of these host and viral proteins in cells and the role of this interaction in replication efficiency. Our results are consistent with a critical role for these interactions in virus biogenesis and pathogenesis for influenza virus infection in both humans and ferrets providing novel anti-viral therapeutic targets for antiviral development
