To investigate the effect of Ras analog in brain 5(Rab5)on the replication of avian metapneumovirus type C(aMPV/C),the recombinant plasmids of pGFP-Rab5 or dominant negative mutant Rab5(pGFP-RAB5-DN)were transfected into A549 cells and inoculated with aMPV/C(MOI 1)24 hours post-transfection,respectively.Confocal microscopy was performed 48 hours post-infection(hpi)to visualize the localization of proteins.The results showed that both Rab5 and Rab5-DN,along with the aMPV/C N proteins,were predominantly localized in the cytoplasm.Notably,co-localization fluorescent signals of Rab5 and aMPV/C N protein were found in Rab5-positive cells,compared to those expressing Rab5-DN.To quantify this interaction,Image J software was utilized to analyze fluorescence intensities along specific lines,confirming multiple points of co-localization between Rab5 and N proteins,with minimal co-localization observed between Rab5-DN and aMPV/C N proteins.These results indicate that Rab5 is involved in aMPV/C replication.Then,further experiments were conducted by transfecting A549 cells with plasmids expressing either Flag-tagged Rab5 or a control vector 24 hours prior to aMPV/C infection.Western blot was employed to measure the relative expression level of the N protein,while fluorescence quantitative PCR(qPCR)was used to assess the transcription level of the aMPV/C N gene.Viral titers were determined using the half tissue culture infective dose(TCID50)method.The results showed that overexpression of Rab5 led to a significant increase in the expression and transcription levels of the aMPV/C N protein and gene,as well as higher viral titers(P<0.01,P<0.05),compared to the control group.Conversely,the knockdown of Rab5 using small interfering RNA(siRab5)resulted in a significant decrease in the expression and transcription levels of the aMPV/C N protein and gene,along with reduced viral titers(P<0.01,P<0.05),when compared to a non-targeting control siRNA(siNC).Co-transfection experiments with plasmids expressing mCherry-tagged Rab5 and GFP-tagged aMPV/C proteins in A549 cells showed that Rab5 co-localized with multiple aMPV/C proteins in the cytoplasm,with the exception of the L4 protein.To validate the interaction between Rab5 and specific aMPV/C proteins,co-transfection was also performed in HEK293T cells.Immunoprecipitation(IP)at 48 hours post-transfection revealed specific interactions between Rab5 and the aMPV/C G and L2 proteins.In summary,Rab5 affects aMPV/C replication by interacting with multiple viral proteins(G and L2).The relevant results provide the basis for elucidating the mechanism of Rab5 regulating aMPV/C replication by interaction.
Rab5avian metapneumovirus type Creplicationexpression levelinteraction
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