A Preliminary Study On The Interaction Between Avian Influenza Matrix Protein M1 And The Host Cell

Influenza virus matrix protein M1, the bridge between the viral envelope and the core with lipid ribonucleoprotein, plays a crucial role in viral morphogenesis. As a major structural component of virus, the M1 protein of influenza virus has multiple regulatory functions in mediating nuclear export of viral ribonucleoproteins, inhibiting viral transcription and virus assembly and budding during the infectious cycle. To illustrate pathogenesis mechanism of avian influenza virus, we find out the target proteins of matrix protein M1 as well as the interaction between proteins using mass spectrometric analysis and yeast two hybridization. Together, these findings shed a light on developing specific targetable therapeutic strategies for antiviral medicine.Firstly, we cloned and expressed matrix protein M1 of Avian influenza virus and obtained high purified M1 protein (86.4% by TLC) by using the Ni2+ affinity chromatography. The surface plasmon resonance biomolecular interaction analysis (SPR/BIA) was used to find out the target proteins binding M1 protein in BHK-21 cell. Results showed the Myosin-6 interacted with M1 protein by the technique of MALDI-TOF MS .Secondly, to find out suspect proteins interacted with the viral M1 protein, the yeast two hybrid systems were used to screen the target gene from cDNA expression library. The M1-interacting cDNA clones were isolated and identified through the yeast interaction trap. After sequencing analysis, four proteins are identified for interacting with M1, they are Clusterin, RAS, F5B(Eukaryotic Translation Initiation Factor 5B)and hypothetical protein.Finaly, the RAS protein was chosen as the research object in the signaling pathway.The interaction between RAS and M1 was confirmed in vivo and in vitro respectively by using co-immunoprecipitation, cell co-localization and BiFC assay.Our results demostrated that the Myosin-6 could interact with M1 protein in vitro, and interacts between M1 Myosin-6 can be inhibited by the formation of polymer Myosin-6. So the Myosin-6 which participates in sweeny, heart regulation, organelle moving and signal transduction pathways, which lead to the basic biochemical functional disorder. By using the yeast two hybrid systems, we assigned four proteins which interacted with M1. Furthermore, the protein of RAS interacting with M1 takes part in the signaling pathway. All above results provide the basis for studying the influenza virus pathogenicity and developing the target for antiviral drugs basing on cellular proteins.