Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells.
The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.
Lee
Meng-Shiou
MS
Graduate Institute of Biotechnology and Center for Nanoscience and Nanotechnology, National Chung Hsing University, Taichung, Taiwan 40227 ROC.
Doong
Shyue-Ru
SR
Lai
Su-Yuan
SY
Ho
Jin-Yi
JY
Wang
Min-Ying
MY
D000818:Animals; D002213:Capsid / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D007243:Infectious bursal disease virus / Q000737:chemistry* / Q000378:metabolism; D020815:Polyproteins / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015678:Viral Structural Proteins / Q000096:biosynthesis*
D020815:Polyproteins; D011994:Recombinant Proteins; C082513:VP2 protein, infectious bursal disease virus; C478244:VP3 protein, infectious bursal disease virus; C478245:VP4 protein, infectious bursal disease virus; D015678:Viral Structural Proteins