EXPRESSION OF TOBACCO RINGSPOT VIRUS CAPSID PROTEIN AND SATELLITE RNAIN INSECT CELLS AND 3-DIMENSIONAL STRUCTURE OF TOBACCO RINGSPOT VIRUS-LIKE PARTICLES

The capsid protein gene of tobacco ringspot virus (TobRV), which had b een modified to contain an amino-terminal methionine codon, was ligate d into a baculovirus transfer Vector downstream from the polyhedrin pr omoter. The resulting plasmid was cotransfected with linearized baculo virus DNA into insect cells. Recombinant baculovirus expressed high le vels of the TobRV capsid protein that assembled to form virus-like par ticles 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, wh ich may lack a putative encapsidation signal. To determine whether an intact packaging substrate could be encapsidated by the TobRV capsid p rotein, 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 circ ular RNA molecules. Like circular forms of satellite RNA generated in planta, these circular satellite molecules remained unencapsidated by the TobRV capsid protein. Computer-generated three-dimensional reconst ruction using electron cryomicrographs of the empty virus-like particl es allowed the first structural analyses of any nepovirus capsid. This 22-Angstrom resolution reconstruction resembled capsids of other memb ers of the picornavirus superfamily. These data support the hypothesis that the nepovirus capsid is structurally analogous to those of the c omo- and picornaviruses. (C) 1995 Academic Press, Inc.