Expression, purification and characterization of enterovirus-71 virus-like particles.
OBJECTIVE
Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region.


METHODS
The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses: Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-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 correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. 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 dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling.


CONCLUSIONS
Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
Chung
Yao-Chi
YC
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, China.
Huang
Jen-Huang
JH
Lai
Chia-Wei
CW
Sheng
Heng-Chun
HC
Shih
Shin-Ru
SR
Ho
Mei-Shang
MS
Hu
Yu-Chen
YC
D000818:Animals; D002460:Cell Line; D004196:Disease Outbreaks; D004770:Enterovirus / Q000145:classification* / Q000302:isolation & purification* / Q000502:physiology; D004769:Enterovirus Infections / Q000453:epidemiology; D006801:Humans; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics
D014764:Viral Proteins