Expression of Tobacco Ringspot Virus Capsid Protein and Satellite RNA in Insect Cells and Three-Dimensional Structure of Tobacco Ringspot Virus-like Particles

Abstract

The capsid protein gene of tobacco ringspot virus (TobRV), which had been modified to contain an amino-terminal methionine codon, was ligated into a baculovirus transfer vector downstream from the polyhedrin promoter. The resulting plasmid was cotransfected with linearized baculovirus DNA into insect cells. Recombinant baculovirus expressed high levels of the TobRV capsid protein that assembled to form virus-like particles that were similar in size and shape to authentic TobRV capsids. These virus-like particles did not encapsidate any RNA, including the capsid protein mRNA. The capsid protein mRNA is a truncated RNA 2, which may lack a putative encapsidation signal. To determine whether an intact packaging substrate could be encapsidated by the TobRV capsid protein, another recombinant baculovirus, concomitantly expressing both capsid protein and TobRV satellite RNA, was constructed. Surprisingly, the vast majority of the satellite RNA molecules expressed from this recombinant baculovirus were ligated in the insect cells to form circular RNA molecules. Like circular forms of satellite RNA generatedin planta,these circular satellite molecules remained unencapsidated by the TobRV capsid protein. Computer-generated three-dimensional reconstruction using electron cryomicrographs of the empty virus-like particles allowed the first structural analyses of any nepovirus capsid. This 22-Å resolution reconstruction resembled capsids of other members of the picornavirus superfamily. These data support the hypothesis that the nepovirus capsid is structurally analogous to those of the como- and picornaviruses.