Function Analysis Of VP466-Rab-tropomyosin-actin Complex In Shrimp Immunity And White Spot Syndrome Virus (WSSV) Infection

White spot syndrome virus (WSSV) is one of the major pathogens in the worldwide shrimp aquaculture. The paper was started at two aspects including virus and host, and the important role of VP466-Rab-tropomyosin-actin complex in shrimp immunity and in WSSV infection was emphatically studied. Our results will not only provide strong theoretical basis for finding WSSV membrane repcepor in shrimp but also be helpful for shrimp disease control.Firstly, the VP466-Rab-tropomyosin-actin complex was identified in this paper. According to the results of GST pull-down and mass Spectrometry assays, the Rab protein was interacted with VP466 of WSSV, shrimp tropomyosin and actin. The interactions among VP466, Rab, tropomyosin and actin were confirmed in vitro and in vivo. The results showed that Rab and tropomyosin proteins were bound with the same domain of VP466 protein.Secondly, the paper was concerned on the functional identification of the VP466-Rab-tropomyosin-actin complex. It was found that the VP466-Rab-actin complex might be involved in shrimp immune response. The gene silencing assays demonstrated that the phagocytic activity of hemocytes was decreased and the WSSV copies were increased when the expression of Rab was inhibited by RNAi. On the other hand, the overexpression results indicated that the the phagocytic activity of hemocytes was increased and the WSSV copies were decreased when the Rab was overexpressed by injection of Rab mRNA. It was found that the Rab GTPase activity was up-regulated by VP466 protein which was investigated in vitro and in vivo. Therefore, one of the putative passways between virus and host immunity was that the interaction between VP466 and Rab-actin could increase the Rab GTPase activity, leading to the increase of phagocytic activity of shrimp hemocytes and the simultaneous descrease of the virus copies. Regarding the role of the VP466-tropomyosin-actin complex, it was shown that the complex was helpful for virus infection. When the VP466 gene was silenced by sequence-specific siRNA, real-time PCR assays suggested that the VP466 protein was an essential membrane protein during the WSSV infection. After the transcription and expression of tropomyosin gene was inhibited by siRNA, real-time PCR and quantitative PCR results revealed that the virus copies was decreased in shrimp. Therefore, one of the putative passways of virus infection was that the interaction of VP466 proein with shrimp cytoskeleton proteins tropomyosin and actin was helpful for virus infection by cytoskeleton motility.Thridly, based on VP466 deletions, it was found that the Rab and tropomyosin proteins were bound with the same domain of VP466. The data suggested that there might be a competition between host immunity and virus infection when the VP466 protein was bound with Rab or tropomyosin.In a word, this was the first report that the WSSV membrane protein VP466 was a double-function protein.