Rab10对禽偏肺病毒C型复制影响的研究OA北大核心CSTPCD

To investigate the effect of Rab10 on the replication of avian metapneumovirus type C(aMPV/C),the recombinant plasmids of pCMV-GFP-Rab10 were transfected into A549 cells and inoculated with aMPV/C before conduct confocal imaging.The results showed that multiple co-localized fluorescent signals of Rab10 and aMPV/C were observed in the cytoplasm while hardly any co-localization signals or aMPV/C signals were found in the mock group.The co-localization coefficients results which were ana-lyzed by Image J software showed that Rab10 andNproteins had significantly higher co-localization co-efficients when compared to the mock group.Furthermore,the plasmids pCMV-Flag-Rab10 and pCMV-Flag as well as siRab10 and siNC sequences were transfected into A549 cells before aMPV/C infection,respec-tively.The supernatural and cell samples were collected at 48 hours post-infection.The transcription level of aMPV/C N gene,the expression level of aMPV/CN and Rab10 proteins,and the viral titers were de-tected by quantitative real-time PCR(qPCR),Western-blot,and the half tissue culture infective dose(TCID50)method,respectively.The results showed that the transcription level of the N gene,the expres-sion level of Rab10 and N proteins,and the viral titers were significantly increased(P＜0.05)after overexpression of Rab10,respectively.On the contrary,the transcription level of the N gene,the ex-pression level of Rab10 and N proteins,and the viral titers were significantly decreased(P＜0.05)af-ter knockdown expression of Rab10,respectively.The above results indicate that Rab10 functions on the replication of aMPV/C.Then,the plasmids of pCMV-mcherry-Rab10 and GFP-tagged aMPV/C proteins were co-transfected into A549 cells,respectively.Obvious co-localized fluorescence signals of Rab10 and aMPV/C proteins(N,F,M,G,SH,P,M2-1,M2-2,L1,L2,and L3)were found in the cytoplasm 24 h post-transfec-tion,while scarcely co-localization signals were found in Rab10 protein and L4 protein co-transfected cells.Subsequently,the plasmids of pCMV-Flag-Rab10 and GFP-tagged aMPV/C proteins were transfected into HEK293T cells before collecting cell samples 36 h post-transfection and conducting the co-immuno-precipitation assay.The results showed that specific bands of P and M2-1 proteins were found in IP sam-ples while no bands of other GFP-tagged aMPV/C proteins were found.These results indicated that Rab10 interacts with aMPV/C P and M2-1 proteins.In summary,Rab10 affects aMPV/C replication by interacting with multiple viral proteins(P and M2-1).The relevant results can provide the basis for elucidating the mechanism of Rab10 regulating aMPV/C replication by interaction.