Studies On The Molecular Mechanism Underlying Avian Reovirus P10 Degradation In Host Cells

Avian reovirus(ARV)is an important pathogen of birds that can cause many clinical diseases,such as viral arthritis,chronic respiratory diseases,retarded growth and malabsorption syndrome.ARV infection also causes immunosuppression,which increases the risks of other diseases,leading to considerable losses to the poultry industry.The ARV p10 protein,a nonstructural protein encoded by the first open reading frame of the S1 gene,is a type-? transmembrane protein and an important virulence factor.The major function of p10 is drilling in the cell membranes,destorying the integrity of cell membranes.P10 induces cell-cell fusion and then promotes ARV reproduction.However,p10 is rapidly degraded in host cells.We previously found that Lysosome Associated Membrane Protein(LAMP-1)interacts with p10 and p10 is degraded via proteasomal pathway.However,how does LAMP-1 play a role in p10 degradation and the molecular mechanism of p10 degradation is still unclear.In this study,we confrimed the interaction between ARV p10 and E3 ubiquitin ligase Siah-1 by immunoprecipitation and laser confocal microscopy approaches,and determined that the N terminal(1aa-43aa)of p10 interacted with Siah-1 while the C-terminal portion(141 aa-282aa)of Siah-I served as the binding domain for p10.Overexpression of Siah-1 significantly reduced p10 levels in DF-1 cells and the reduction of plO by Siah-1 could be markedly inhibited by proteasome inhibitor MG132,indicating that p10 degradation dependents on ubiquitin-proteasome system.Knockdown of endogenous Siah-1 by RNAi enhanced p100 levels.Overexpression of Siah-1 enhanced p10 ubiquitylation while knockdown of Siah-1 by RNAi markedly reduced the ubiquitylation of p10,demonstrating that Siah-1 promotes p10 ubiquitylation.Furthermore,Siah-1 could mediate p10 ubiquitylation in vitro,Overexpressin of Siah-1 in DF-1 cells promoted ARV growth.On the contrary,knockdown of Siah-1 enhanced ARV growth and increased the amount of ARV-induced syncytium.This discovery indicated that Siah-1 plays a key role in ARV reproduction.The co-immunopreciptation experiments in ARV infected DF-1 cells revealed that p10 interacts with both Siah-1 and LAMP-1 and they formed a complex.Knockdown of LAMP-1 by RNAi markedly reduced ARV p10 in the precipitates from the lysates immunoprecipitated by Siah-1 and decreased the colocalization of Siah-1 with p10.Meanwhile,LAMP-1 RNAi inhibited the reduction of p10 by Siah-1 overexpression.This result shows that Siah-1 promotes p10 ubiquitylation and degradation via interaction with LAMP-1 and p10.In summary,our results demonstrate Siah-1 promoted p10 degradation through facilitating p10 ubiquitylation and surpresses ARV reproduction.LAMP-1,via interaction with both Siah-1 and p10,serves as a scaffold protein that allows the E3 ligase targeting p10 for its ubiquitylation and degradation,and suppresses viral growth.