The Molecular Mechanism Underlying ARV Sigma-C-induced Apoptosis In Host Cells

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome, leading to considerable losses to the poultry industry. ARV-induced apoptosis plays an important role in tissue injury caused by ARV infection. It is well established that, among the viral proteins of ARV, sigma-C protein could induce apoptosis in host cells. However the underlying molecular mechanism for such induction remains unclear.In this study, the datas of flow cytometry showed that ARV σC could induce apoptosis in DF-1(immortal chicken embryo fibroblast), BHK-21(Baby Hamster Syrian Kidney) and Hela (Henrietta Lacks strain of cancer cells) cells. To further investigate the molecular mechanism underlying aC-induced apoptosis, we performed Yeast-two hybrid assay with σC as the bait to screen the chicken spleen cDNA library and used colony-lift filter assay to confirm the interaction.To facilitate this study, we developed monoclonal antibodies using recombinant aC as an immunogen by hibridoma technique and get two strains of hybridoma that can stably secret monoclonal antibodies against VC, and both two McAbs have high affinity and specificity. The datas of Western Blot and IFA (Immunofluorescence antibody assay) showed that the obtained McAbs could specificly recognize aC protein in ARV-infected cells, and be effectively used in the followed study.Among the positive clones, eukaryotic elongation factor] alpha1(EEF1A1) was identified as an interacting partner with aC in yeast and selected for further analysis in association with apoptosis. We found that σC interacted with EEF1A1in host cells by coimmunoprecipitation and confocal laser scanning microscopy assays, and determined the domain that spanning residues210-246of σC was involved in interacting with EEF1A1by pulldown assay. Importantly, we found that knockdown of endogenous EEF1A1could completely abolish σC-induced apoptosis but partly inhibit ARV-induced apoptosis in DF-1cells. Furthermore, knockdown of EEF1A1markedly decreased aC/ARV-induced Caspase-9and Caspase-3activations and cytochrome C release, and leaded to increased ARV growth in host cells.In summary, EEF1A1plays a critical role in σC-induced apoptosis and inhibition of viral growth, and these findings help to elucidate the pathogenesis mechanism of ARV infection and provide an important clue to the development of novel vaccines and the control of this disease.