The Mechanism Of ARV Entering Cells Via Caveolin-1-Dependent Endocytic Pathway

Avian Reovirus(ARV)is an important pathogen that may cause considerable economic losses in the poultry industry.ARV has been implicated in several severe diseases in domestic fowl,such as viral arthritis/tenosynovitis,chronic respiratory disease,runtingstunting syndrome and immunosuppression.In this study,we found that ARV enters cells via caveolin-dependent endocytosis,and the σB protein of ARV interacts with Caveolin-1 in lipid rafts during this process.It may help to understand virus-host interactions during the entry routes of ARV,and provide new insights into the pathogenesis and control of ARV.1.ARV entry involves Caveolin-dependent endocytosisPurified ARV virions were generated using sucrose gradient centrifugation.Vero and DF-1 cells were infected with the purified ARV virions at a multiplicity of infection(MOI)of 50 for 30 min at 37℃,and cells were collected and processed for ultrathin sections.The caveolar invaginations on the cell membrane and the virus-containing caveolar vesicles were observed under transmission electron microscopy.To investigate the entry routes of ARV,specific inhibitory drugs were used to evaluate the importance of dynamin,caveolae,and clathrin during ARV entry.The cells were treated with the indicated concentrations of dynasore,CPZ or nystatin for 1 h at 37℃ prior to infection of ARV.The efficiency of ARV internalization was evaluated by RT-qPCR and western blotting at 12 hpi.Besides,the cells in each group were stained with Giemsa at 12 hpi for syncytium formation assay.We found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors,while it is insensitive to clathrin inhibitors.In conclusion,these results indicate that the ARV entry involves caveolin-dependent endocytosis.2.ARV σB protein interacts with Caveolin-1In this study,the membrane lipid rafts were isolated as detergent-resistant microdomains(DRMs)by sucrose gradient centrifugation and the capsid protein σB of ARV was found to associate with Cavelin-1 in caveolar lipid rafts at 1 hpi and 12 hpi.Additionally,the ARV protein p10,p17,σNS were presented in DRMs contained the lipid rafts microdomains at 12 hpi.In conclusion,these results suggest that the caveolar lipid rafts of the cell membrane are involved in the entry of ARV.Furthermore,the expression plasmids pCDNA3.1-σB-Flag and pCDNA3.1-Cav-1-MyC were constructed and co-transfected into Vero and DF-1 cells.The interaction between ARVσB protein and Caveolin-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays.To further confirm our findings,siRNA was designed and synthesized to specifically knock down of Caveolin-1.Compared with the presence of control siRNAs,expression of Caveolin-1 in Vero and DF-1 cells transfected with Caveolin-1 siRNA significantly reduced.By silencing caveolin-1,a significant reduction in the mRNA levels of the μNS and σC genes of ARV was presented in both Vero and DF-1 cells.Altogether,these results suggest that Caveolin-1 plays an important role in the replication of ARV.