Different architectures in the assembly of infectious bursal disease viruscapsid proteins expressed in insect cells

Infectious bursal disease virus (IBDV) capsid is formed by the processing o f a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the corr ect assembly of the viral capsid in vitro, different constructs were made u sing two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, t he expression of the capsid proteins gave rise to different types of partic les in each system, as observed by electron microscopy and immunofluorescen ce. FastBac expression led to the production of only rigid tubular structur es, similar to those described as type I in viral infection. Western blot a nalysis revealed that these rigid tubules are formed exclusively by VPX. Th ese tubules revealed a hexagonal arrangement of units that are trimer clust ered, similar to those observed in IBDV virions. In contrast, pAcYM1 expres sion led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of I BDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only o n the inner surfaces of VLPs and caps of the tubular structures. In summary , assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. (C) 2000 Academic Press.