The Molecular Mechanism Underlying ARV P10-induced Apoptosis And Its Degradation In Host Cells

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, malabsorption syndrome and immunosuppression. The ARV p10 protein, a nonstructural protein encoded by the first open reading frame of the S1 gene, is a type-I transmembrane protein. It is reported that p10 is responsible for the induction of cell syncytium formation and apoptosis, and is rapidly degraded in host cells. However the mechanism underlying P10 activities in infected cells are still unclear.In this study, we analyzed the mechanism of p10 activities in ARV s1133 infected DF-1 cells. We cloned p10 gene and made pEGFP-p10 construct, and transfected cells with the constructs, followed by analysis of apoptosis with flow cytometry. We found that p10 could induce apoptosis in DF-1 cells. To search for the target protein interacting p10, we employed yeast two-hybrid screen system using p10 as a bait to screen a cDNA library generated from the chicken spleen. We found that LAMP-1 interacted with p10 in yeast two-hybrid screening. We further conformed the interaction of p10 with LAMP-1 in BHK-21 and DF-1 cells via coimmunoprecipitation. Using confocal microscopy assays, we found that ARV p10 and LAMP-1 were colocalizedin cells, and they were located in lysosome.Our data show that the half-life of p10 in cells was very short. To investigate how p10 is degradation in cells, we cultured cells in the presence of the proteasome inhibitor MG132 and examine the half-life of p10. As a result, we found that the rapid degradation of p10 could be inhibited by MG132, indicating that the degradation of P10 was related to proteasomal pathway. Since degradation of proteins via proteasomal pathway required ubiquitination, we examined the ubiquitination p 10. As a result, p10 was associated with K63-and K48-linked ubiqintination, suggesting that p10 was degraded via the ubiquitination-dependent proteasomal pathway. The fact that p10 induces apoptosis, subjects to ubiquitin-proteasomal degradation, and interacts with LAMP-1 suggests that LAMP-1 plays a role in p10-induced apoptosis and/or in its degradation, and that knockdown of LAMP-1 would therefore affect apoptosis and/or degradation of p10. To test this hypothesis, we made three LAMP-1 RNAi constructs and found that one of them could effectively lower the cellular level of LAMP-1 without causing discemable changes in cell morphology. Then we transfected cells with both pRK5-flag-p10 and HA-linked ubiquitin (HA-Ub) expression vectors in the presence of MG132.We found that the degradation and ubiqintination of p10 in host cells could be completely abolished by knockdown of LAMP-1 by siRNA. Furthermore, Knockdown of LAMP-1 allows p10 accumulation, enhancing p10-induced apoptosis and viral release. In contrast, overexpression of LAMP-1 markedly reduced p10 in cells and this reduction was shown in dose-dependent manner.In summary, our results demonstrate that ARV p10 can induce apoptosis in DF-1 cells, and further show that p10 interacts with avian LAMP-1. These data indicate that LAMP-1 plays a critical role in ARV p10 degradation by promoting the ubiqintination of p10 and associates with inhibition of apoptosis and viral release. These findings have provided insights for elicitation of the mechanism underlying p10-induced apoptosis and its degradation and furthered our understandings of the pathogenesis of ARV infection and development of vaccine.