Assembly of the T = 4 Nudaurelia capensis omega virus capsid protein, post-translational cleavage, and specific encapsidation of its mRNA in a baculovirus expression system

Abstract

We have expressed the gene encoding the coat protein (CP) of Nudaurelia capensis omega virus in insect cells with a baculovirus vector. Expression of CP resulted in formation of virus-like particles (VLPs) having a size consistent with the T = 4 quasi-symmetry observed for native virions. This is the first demonstration of assembly for a T = 4 particle, with chemically identical subunits present in four distinct environments, by heterologous expression. Initial yields of VLPs were low, and an efficient one step nondenaturing procedure involving separation on discontinuous glycerol gradients was developed. Using this method, VLPs were obtained in quantities sufficient for further characterization. Electron microscopic observation revealed 40-nm particles that were morphologically similar to native virus. SDS-PAGE revealed that these particles were composed of a 62-kDa major protein and a minor 70-kDa protein. Pulse-chase experiments revealed that the larger species was processed into the smaller one very slowly over the course of an infection. It was also determined that this cleavage was apparently dependent on release of these particles from the cell. Furthermore, these particles were found to encapsidate the polyhedrin promoter-directed CP mRNA with an apparently striking degree of specificity and selectivity. This investigation establishes that a specific encapsidation signal exists within the CP coding sequences and that the components required for reconstructing most, if not all, steps in the morphogenesis of this virus can be accomplished in baculovirus-infected cells. The results presented here are consistent with the belief that similar biological strategies are utilized by T = 3 nodaviruses and T = 4 tetraviruses in particle assembly.