Effect of Rab32 on the Replication of Avian Metapneumovirus Type C
To investigate the effect of Rab32 on the replication of avian metapneumovirus type C(aMPV/C),confocal images were obtained and co-localization coefficients were analyzed by Image J software after infected with A549 cells by aMPV/C.Furthermore,the plasmids of pCMV-Flag-Rab32 or pCMV-Flag as well as siRab32 or siNC RNA sequences were transfected into A549 cells before aMPV/C infection,respectively.The transcription level of aMPV/C N gene,the expression level of aMPV/C N and Rab32 proteins,and the viral titers in collected samples were detected by quantitative real-time PCR(qPCR),Western blot,and the half tissue culture infective dose(TCID50)method,respectively.Then,the plasmids of pCMV-mcherry-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into A549 cells before conducting confocal imaging,respectively.Subsequently,the plasmids of pCMV-Flag-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into HEK293T cells before collecting cell samples at 36 h post-transfection and conducting the co-immunoprecipitation assay.The results showed that obvious co-localized fluorescent signals of Rab32 and aMPV/C were observed in the cytoplasm.The co-localization coefficients of Rab32 and N proteins were significantly higher than that of mock-infected group.The transcription level of the N gene,the expression level of Rab32 and N proteins,and the viral titers were significantly increased(P＜0.01)after overexpression of Rab32,respectively.On the contrary,the transcription level of the N gene,the expression level of Rab32 and N proteins,and the viral titers were significantly decreased(P＜0.01)after knockdown expression of Rab32,respectively.Obvious co-localized fluorescent signals of Rab32 and aMPV/C proteins(N,F,M,G,SH,P,M2-1,M2-2,L1,L2,and L4)were observed in the cytoplasm,while scarcely co-localized signals of Rab32 and L3 protein were found.Specific bands of N and M2-2 proteins were found in immunoprecipitation samples while no bands of other GFP-tagged aMPV/C proteins were found.These results indicating that Rab32 interacts with aMPV/C N and M2-2 proteins.In summary,Rab32 might affect aMPV/C replication by interacting with multiple viral proteins(N and M2-2).The relevant results can provide the basis for elucidating the mechanism of Rab32 regulating aMPV/C replication via protein interaction.
Rab32avian metapneumovirus type creplicationexpression levelinteraction
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维普
