Identification Of Tiger Frog Virus (TIF) Host-derived Protein Caveolin1

ABSTRACTTiger Frog Virus (TFV）， member of Iridoviridae， has a wide host range. It’sone of the reasons that cause the death of amphibian， reptiles and fish. And it hasbeen known that virions contain host-derived proteins (HDPs） in addition to proteinsencoded by the viral genome. In the thesis， we identified HDPs from TFV and thelocation of caveolin1in TFV virion and explained how TFV incorporates caveolin1.Firstly，TFV virions are highly purified from infected HepG2cells by CsCldensity gradient centrifugation and infectious. Based on the shortgun results and TFVintrusion mechanism， we analysed the potential HDPs by western blotting and foundthat TFV virions incorporate caveolae involved proteins including caveolin1，caveolin2， cavin1and cavin2and cytoskeletal proteins like β-actin and filamin butclathrin and flotillin. And HepG2cells were transfected by recombinant plasmidcaveolin1with flag tag (caveolin1-flag) and then infected TFV. Western blotting candetect caveolin1-flag protein in TFV purified from the transfected HepG2cells，which proved that TFV virions specifically incorporate caveolin1. By electronmicroscopy and western blotting， we found extracted caveolae contain TFV virions， suggesting that caveolae and TFV virions are closely related and TFV virions mayincorporate caveolae involved proteins.After the enzyme and chemical treatment， caveolin1is identified located insideTFV capsid and probably face to the virion core. Caveolin1is proved to interact withTFV major capsid protein (MCP) by co-immunoprecipitation and immunolocalization，which illuminated how TFV incorporates caveolin1.At last， we tried to infect cells which are reported to low express caveolin1.Only can MCF-7cells low express caveolin1. But caveolin1expression upregulatesafter MCF-7is infected by TFV. Caveolin1can also be detected in virions purifiedfrom infected MCF-7. As a result， we hope that TFV virions without caveolin1as anegative control can be found from caveolin1knock-out cells， which wills help us tounderstantd the mechanism of HDP caveolin1on TFV. The results will lead us tofurther study the mechanism about HDPs on Iridoviridae intrusion， reproduction，package， budding and immune escape.