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.

Strong and heterogeneous adsorption of infectious bursal disease VP2 subviral particle with immobilized metal ions dependent on two surface histidine residues.

VP2, the single outer protein of infectious bursal disease virus capsid, can self-assemble into T = 1 subviral particle (SVP), which can be efficiently purified by immobilized metal ion affinity chromatography (IMAC). In this study, a systemic investigation of the adsorption behavior of VP2 SVP on Ni-NTA resin was performed to identify that His253 and His249 on the surface of SVP are the key factors accounted for the strong and heterogeneous interaction. First, an untagged VP2-441 SVP was constructed, expressed, and purified by IMAC to demonstrate that SVP can interact with immobilized Ni2+ ions on NTA resin without an inserted His tag. Second, equilibrium adsorption studies were used to demonstrate that SVP has a higher affinity to the immobilized Ni2+ ions than a model protein, bovine serum albumin, although the maximum amount of SVP bound per volume resin is limited by the pore size of the resin as verified by confocal microscopic analysis. Third, based on structural analysis and computer modeling, His253 and His249 on the surface of SVP are responsible for a strong heterogeneous and multiple adsorption with the immobilized Ni2+ ions; and this was confirmed by a point-mutation experiment. This is the first example to elucidate the interaction between the immobilized metal ions and viral particles at molecular level. A detailed understanding of SVP-immobilized metal ion interactions can provide useful strategies for engineering icosahedral protein nanoparticles to achieve a simple and one-step purification by IMAC.

Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity.

The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes.