Unveiling the neglected roles of nucleoprotein NLS2 and cellular vimentin during Influenza A virus infection

Influenza A virus exploits the cellular transport machinery during the early stages of infection. It enters cells by endocytosis and takes advantage of the endocytic trafficking to move towards the perinuclear region with the assistance of actin filaments and microtubules. A recent proteomic study identified vimentin as a putative interacting protein of influenza viral components. However, the role of vimentin during influenza A infection has not yet been determined. After endocytosis, the viral ribonucleopotein complexes (vRNPs), containing the RNA viral genome, the viral polymerases, and several copies of nucleoprotein, are released from late endosomes and enter the nucleus for replication. Two nuclear localization sequences (NLSs), NLS1 and NLS2, on nucleoprotein mediate the nuclear import of vRNPs. The function of NLS1 has been well studied, however, the role of NLS2 remains to be defined. This thesis has two major aims: to characterize the function of NLS2 and to determine the role of vimentin during influenza infection. For the first aim, I use a chimeric protein (5GFP) fused to NLS2, in combination with RNAi of several importin α isoforms and biochemical assays, and found that NLS2 is able to mediate the nuclear import of 5GFP by interacting with importin α1, α3, α5, and α7. NLS2 contains only a single amino acid difference at position 17 between different strains of influenza A virus, which could be either lysine (K) or arginine (R). I found that NLS2K induces more nuclear accumulation of 5GFP than NLS2R. Using site-directed mutagenesis I demonstrated that NLS2K contains two independent functional basic clusters, while NLS2R only has one. Moreover, my study also revealed that inhibiting the function of NLS1 and NLS2 impairs the nuclear import of vRNPs and further inhibits viral infection. For the second aim I followed influenza A virus infection in both vimentin null cells and vimentin RNAi knock-down cells, and found that vimentin plays a role in releasing vRNPs from endosomes. In summary, my work dissects the basic mechanisms involved in influenza A virus endocytic trafficking and nuclear import, which provide us with better insights into the viral-host interaction during influenza A virus infection.Science, Faculty ofZoology, Department ofGraduat