Influenza Viral Protein Interaction Detecting Methods Development and A.M1-host Interplay Network Investigation

The viral RNA-dependent RNA polymerases of influenza A and B are trimetric complexes (PA, PB1, and PB2 subunits), and are crucial for both transcription and replication of the viral genome. While there are extensive data on the structure and function of influenza A polymerase, the influenza B polymerase has received less attention, despite the incidence of severe influenza B respiratory infections in humans. The aim of this study is to characterize the influenza B RNA polymerase complex subunits interaction. In addition to two protein complementation assays, Split luciferse complementation assay (SLCA), and Bimolecular Fluorescence Complementation (BiFC), LUMIER (luminescence-based mammalian interactome mapping) assay were also developed and used in this study. We demonstrated pairwise interactions among the influenza B subunits (PA-PB1, PA-PB2, and PB1-PB2). Among these interactions, PAPB1 and PB1-PB2 interactions are observed in all three assays and the PA-PB1 interaction is much stronger than the PB1-PB2. A weak and transient interaction between PA-PB2 was detected by SLCA and BiFC assays, but not in LUMIER assay. Furthermore, we showed that each of three influenza B polymerase subunits and associated binary complexes, like its counterpart in influenza A, possesses nuclear import function. This study provides a framework for further characterization of influenza B viral polymerase complex assembling molecular mechanisms and exploitation of this machinery as a target for antiviral discovery. M1 protein, known as a linkage, connects external envelope proteins and internal core proteins, and also plays significant role in virus structure morphology. However, the other functions of M1 has not been defined. In this study, we used a bait-fishing strategy by applying over-expressed 3×Flag-A.M1 as bait to fish potential interacting cellular proteins in 293T cells. 99 unique proteins have been pulled down by 3×Flag-A.M1 compare to the negative control of empty 3×Flag vector and 3×Flag-Luciferase. 99 cellular proteins were identified and several cellular proteins in our results were also discovered by previous researches that interact with M1 protein. Most of cellular proteins are novel to M1-host protein interaction network, after analyzed by immunoprecipitation assay (IPA), seven canonical pathways were found highly relative in over expressed M1 293T cells. Those are protein ubiquitination pathway, actin cytoskeleton signaling pathway, epithelia adherences junction signaling, mechanism of viral exit from host cells signaling, DNA double-strain break repair by non-homologous end joining pathway, aldosterone signaling in epithelia cells and granzyme B signaling. And mechanism of viral exit from host cells, DNA double-stain break repair by non-homologous end joining and granzyme B signaling are top three abundant pathways been identified