Processing of Infectious Bursal Disease Virus (IBDV) Polyprotein and Self-Assembly of IBDV-Like Particles in Hi-5 Cells

Abstract

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.

Chicken Heat Shock Protein 90 Is a Component of the Putative Cellular Receptor Complex of Infectious Bursal Disease Virus

“…In our study, SVP is unique because the size of SVP (ϳ25 nm) is over two times smaller than that of IBDV (ϳ65 nm). Despite the smaller size, the preparation of SVP is much simpler than that of IBDV capsid or IBDV-like particles (virus-like particles) (21). There are two more reasons why SVP was used.…”

“…At 3 to 4 days postinfection, cells were observed for cytopathic effects (CPEs) using an inverted microscope. After being detached with 5 mM EDTA, cells were collected for immunoblotting assays using polyclonal anti-VP2 or anti-VP3 antibodies (21), and the supernatant, in general with a titer of 6 ϫ 10 6 PFU/ml, was saved as a viral stock. Generation of a recombinant baculovirus-expressing IBDV VP2-441-formed SVP.…”

“…Putative recombinant baculoviruses were then plaque purified. The expression of the VP2-441 monomer protein in Sf9 or Hi-5 cells was characterized by Western blotting using a polyclonal antibody, anti-VP2 (21).…”

“…SVP formed by VP2-441 was produced and purified by using a previously established protocol (20). Purified SVP was characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) staining with silver nitrate, Western blotting, and negative-stain electron microscopy (21). For conjugation of SVP with FITC (Sigma), 4 mg of SVP was resuspended in 500 mM carbonatebicarbonate buffer (pH 9.5), mixed with 400 g of FITC, and incubated at room temperature for 1 h (18).…”

“…Following electrophoresis, the gel was stained by silver nitrate or used for Western blotting analysis by electroblotting onto a polyvinylidene difluoride (PVDF) membrane. To detect IBDV antigens (pVP2/VP2 and VP3), the polyclonal anti-VP2 and anti-VP3 antibodies (21), respectively, were employed as the first antibody. To detect cHsp90, either anti-Hsp90 mouse MAb (anti-Hsp90 MAb from Novus Biologicals, Inc., CO) or rat anti-Hsp90␣ MAb (Stressgen Biotechnologies, Victoria, British Columbia, Canada) was used as the primary antibody.…”