The Role Of Rab6Protein In The Antiviral Phagocytosis Of Drosophila Melanogaster S2Cell

Rab GTPase, which is the largest family of small GTP-binding proteins superfamily, can regulate the communication between the organelles. Rab GTPase can interact with regulators and effectors as the molecular switch of endosomes trafficking. However, the role of Rab GTPases in the immune system remains to be further investigated.Based on the previous studies in our lab, it is found that the shrimp Rab6, a member of Rab superfamily, is involved in the regulation of the antiviral phagocytosis of shrimp hemocyte by interaction with actin protein. Both shrimp and the model organism Drosophila belong to arthropoda. Moreover the homology of Rab6protein between the two species is90.47%. Therefore the Drosophila S2cell was used in this study to reveal the mechanism of phagocytosis against the Drosophila C virus (DCV). This would be helpful to get a comprehensive view on the antiviral phagocutosis in invertebrate.In an attempt to reveal the role of Rab6protein in the phagocytosis of fruit fly, the S2cell line which had the phagocytic activity was employed in this investigation. To detect the expression of Rab6in S2, the fruit fly Rab6-specific antibody was prepared. The Western blot showed that the Rab6protein could be detected in S2cells. The previous data of our lab indicated that the Rab6could regulate phagocytosis by interacting with β-actin protein. So the fruit fly Rab6andp-actin recombinant plasmids were co-transfected into S2cells, following by co-immunoprecipitation assays. The results showed that the Rab6could interact with actin in S2, suggesting that Rab6might be involved in phagocytosis by interaction with actin.To determine the function of Rab protein in S2phagoctosis, first, the Rab6gene was silenced, over-expressed and rescued, respectively. When the Rab6-specific siRNA was transfected into S2cells to silence the Rab6gene, the real-time PCR and Western blot assays showed that the Rab6mRNA and protein were significantly decreased. At the same time, the phagocytic activity of S2cells using the FITC-labeled inactivated DCV was significantly decreased in response to the Rab6gene silencing. When the Rab6gene was upregulated by transfection of the recombinant plasmid containing Rab6into S2, the S2phagocytic activity against DCV was significantly enhanced with the upregulation of Rab6. On the basis of the above assays, the Rab6-specific siRNA and the recombinant construct containing the synonymous mutations of Rab6protein to avoid the recognition by Rab6-specific siRNA were co-tansfected into S2cells to inhibit the expression of endogenetic Rab6and to produce exogenetic Rab6protein, respectively. The results showed that the Rab6was rescued in the Rab6-knocked-down S2cells, leading to the recovery of S2phagocytic activity against DCV. The data demonstrated that the Rab6protein could regulate the phagocytic activity of S2cells.To investigate the effects of Rab6-regulated phagocytosis on virus infection, the multiplicity of infection (MOI) of DCV in S2was evaluated in response to the Rab6silencing, over-expression or rescue. The results presented that the MOI in the Rab6-silenced S2cells was higher than that in control and the over-expression of Rab6led to lower MOI. When the Rab6-specific siRNA and the recombinant plasmid containing Rab6were con-transfected into S2cells, it was found that the rescue of Rab6could inhibit the DCV infection. Therefore the Rab6protein could regulate the antiviral phagocytosis.In order to get a visual view of phagocytic process against DCV, the Rab6-actin protein complex was labeled and examined under confocal microscope. The results showed that the Rab6protein was gathered in the sites of phagocytic virions during the S2phagocytosis, suggesting the importance of Rab6protein in the phagocytosis against virus infection.Based on the above data, it was concluded that the Rab6protein could regulate the antiviral phagocytosis in fruit fly.