Rab18调控禽偏肺病毒C型复制的研究OA北大核心CSTPCD

This work aimed to investigate how Rab18 influences the replication of avian metapneu-movirus type C(aMPV/C)in A549 cells.Confocal images were obtained by laser confocal microscope af-ter being infected with aMPV/C.The colocalization coefficient was analyzed by Image J software.The results showed that Rab18 and aMPV/C N protein were expressed in the cytoplasm and showed obvious co-localization,while no fluorescent signal of N protein was observed in the mock group.The co-local-ization coefficient between Rab18 and aMPV/C N protein was significantly higher than that in the mock group(P＜0.01).The plasmids(pCMV-Flag-Rab18 and pCMV-Flag)and nucleic acids(siRab18 and siNC)were transfected into A549 cells before being infected with aMPV/C,respectively.Cell samples and cell su-pernatant were collected for detection of the transcription level of aMPV/C Ngene,the expression lev-el of N protein,and viral titers by qPCR,Western-blot,and TCID50,respectively.The results showed that the transcription level of aMPV/C Ngene,the expression level of N protein,and the titer of virus were significantly increased after overexpression of Rab18,respectively(P＜0.01).On the contrary,the transcription level of aMPV/C Ngene,the expression level of N protein,and the viral titers were sig-nificantly decreased after knockdown expression of Rab18,respectively(P＜0.01).A549 cells were co-transfected with pCMV-mcherry-Rab18 and GFP-tagged aMPV/C genes before confocal imaging.The results showed that Rab18 could co-localize with aMPV/C proteins in the cytoplasm.HKE293T cells were co-trans-fected with pCMV-Flag-Rab18 and GFP-tagged aMPV/C genes before conducting the co-immunoprecipitation(co-IP)assay.The results showed that specific N,M2-1,and L2 protein bands were found in IP samples while other viral proteins were not detected.These results suggest that Rab18 influences aMPV/C replication via interaction with N,M2-1,and L2 proteins.The results provided a foundation for further analysis of the molecular mechanism of regulation aMPV/C replication by Rab18.