Expression,purification and characterization of enterovirus-71 virus-like particles

AIM:Enterovirus 71(EV71)has been implicated as theetiological agent responsible for the recent outbreaksof hand,foot and mouth disease associated with severeneurological diseases in the Asia-Pacific region.METHODS:The assembly process was hypothesizedto occur via an orchestrated proteolytic processing ofthe P1 precursor by the viral protease 3CD.To test thishypothesis,we constructed 3 recombinant baculoviruses:Bac-P1 expressing P1;Bac-3CD expressing 3CD;andBac-P1-3CD co-expressing P1 and 3CD.RESULTS:Both single infection by Bac-P1-3CD and co-infection by Bac-P1 and Bac-3CD resulted in correctcleavage of P1 to yield individual proteins VP0,VP1and VP3,while the former approach yielded higher VLPproduction.In the cells,the structural proteins self-assembled into clusters of virus-like particles(VLP)resembling the authentic EV71 particle aggregates.After ultracentrifugation purification,the dispersedVLPs were indistinguishable from the authentic virus insize,appearance,composition and surface epitopes,asdetermined by SDS-PAGE,Western blot,transmissionelectron microscopy and immunogold labeling.CONCLUSION:Our data,for the first time,suggestthat in insect cells EV71 structural proteins adopt aprocessing and assembly pathway similar to poliovirusassembly.The preservation of particle morphology andcomposition suggest that the VLP may be a valuablevaccine candidate to prevent EV71 epidemics.