Identification And Functional Analysis Of Structural Proteins Of White Spot Syndrome Virus

White Spot Syndrome Virus (WSSV) represents a new genus of DNA viruses, Whispovirus, belonging to the Nimaviridae family, and is a major pathogen in the cultured penaeid shrimp. It has a wide range of hosts in crustaceans, a high infection and mortality rate, and is hard to be controlled. Therefore WSSV is not only a major threat to the shrimp industry but also to the marine environment. In this thesis, a comprehensive proteomic analysis was made to define and characterize the major envelope and nucleocapsid proteins of WSSV. Moreover, the functional exploration and interaction of WSSV structural proteins was investigated.WSSV virions were purified from the tissues of infected crayfish Procambarus clarkii. Pure WSSV preparations were subjected to Triton X-100 treatment to separate into the envelope and nucleocapsid fractions, which were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major envelope and nucleocapsid proteins were identified by either matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or defined antibody. A total of 30 structural proteins of WSSV were identified in this study; 22 of these were detected in the envelope fraction, 7 in the nucleocapsid fraction and 1 in both the envelope and the nucleocapsid fractions. With the aid of specific antibodies, the localization of 8 proteins was further studied. Moreover, the analysis of post-translational modifications revealed that none of WSSV structural protein was glycosylated and acetylated, and VP28 and VP19 were threonine-phosphorylated.VP26, the product of the wsv311 gene of WSSV, is one major structural proteins of virus. When purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as Actin by mass spectrometry (MS). Moreover, the association of VP26 protein with Actin microfilaments was confirmed by coimmunoprecipitation.VP24, the product of the wsv002 gene of WSSV, is one major structural protein of virus and thought to be a nucleocapsid protein in the previous report. In this study, a more precise localization of VP24 within WSSV virions was carried out. When purified virions were subjected to Triton X-100 treatment to separate the envelopes from the nucleocapsids, VP24 was found to present exclusively in the envelope fraction. Triton X-114 extraction also indicated that VP24 behaves as an envelope protein. Immunoelectron microscopy further confirmed that VP24 is located in the envelope of virion. To investigate the function of VP24, WSSV was incubated with various amount of anti-VP24 IgG and injected into crayfish. The result showed that anti-VP24 IgG could partially attenuate infection with WSSV. We concluded that VP24 is an envelope protein and functions at an early stage in virus infection.Finally, interactions between envelope proteins and between viral envelope proteins with cellular membrane proteins were investigated. Far-Western and coimmunoprecipitation experiment showed that VP28 interacted with both VP26 and VP24. In addition, an envelope-protein-binding protein with an apparent molecular mass of 22 kDa were purified from membrane of crayfish hemocytes by affinity-chromatography and identified as a cAMP receptor protein by MS. In summary, the data obtained in this study should provide an important reference for future molecular studies of WSSV morphogenesis.