Resumen de Bases estructurales de la cápsida del virus de la bursitis infecciosa para el desarrrollo de futuras aplicaciones biotecnológicas
Infectious bursal disease virus (IBDV) is a polyploid, bisegmented dsRNA virus with a single, non-enveloped ~70-nm-diameter capsid with a T=13l lattice. The capsid structural units are trimers of a single protein, VP2 (441 residues). The VP2 precursor form (pVP2) is encoded as part of the polyprotein NH2-pVP2-VP4-VP3-COOH; VP4 is the viral protease, able to cleave its own N and C termini, and VP3 is a multifunctional protein with scaffolding and dsRNA-binding activities. The pVP2 71-residue C-terminal domain, which is proteolytically processed, bears the molecular switch that controls VP2 polymorphism, the 443GFKDIIRAIR452 amphipathic ¿-helix. In the absence of VP3, pVP2 intermediates with this ¿ -helix assemble into genuine virus-like particles (VLP) only when expressed with an N-terminal His tag (chimeric protein HT-VP2-466).
T=13 HT-VP2-466 capsids are optimal for insertion of heterologous proteins, as they are very spacious (~77,900 nm3 cargo space). We explored new HT-VP2-based chimeric capsids, using the enhanced green fluorescent protein (EGFP) fused at its N-terminal end as the initial model. As EGFP-HT-VP2-466 expression in a recombinant baculovirus system is inefficient for VLP assembly, we improved VLP yield by coexpressing EGFP-HT-VP2-466 and HT-VP2-466 proteins from two recombinant baculoviruses at various ratios; larger amounts of EGFP-HT-VP2-466 protein were assembled as VLP. Stoichiometric analysis estimated that ~240 EGFP-HT-VP2-466 copies/virion are incorporated. EGFP is incorporated with a native structure, as VLP are fluorescent. Three-dimensional cryo-electron microscopy of EGFP-containing VLP showed that EGFP molecules are located at the inner capsid surface. Chimeric EGFP-VLP particles also elicit antibodies to native EGFP when mice are immunized with purified particles.
As these chimeric VLP are excellent ¿nanoboxes¿ for carrying macromolecules to living cells, other epitopes of interest derived from the murine influenza virus A/PR8 were inserted. We selected fragments of hemagglutinin (HA) and matrix (M2) proteins, both of which elicit antibodies with broad neutralizing activity. Several HA- and M2-derived chimeric capsids were used to immunize mice. Mice immunized with VLP containing HA2 (the stalk subunit), an M2 fragment, or HA2 and an M2 fragment simultaneously, produced antibodies that recognized murine influenza virus A/PR8 and/or the M2 peptide, and were fully protected against viral challenge.
Finally, we established an in vitro disassembly/self-assembly system of T=13 VLP to generate capsids similar to virions. HT-VP2-466 (empty) capsids can be disassembled and reassembled by dialysis. No previous reports indicated the reversibility of the IBDV assembly process. Furthermore, HT-VP2-466 VLP can encapsidate heterologous DNA by non-specific confinement during assembly.
These results establish a basis for future biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.
