EXPRESSION OF TOBACCO RINGSPOT VIRUS CAPSID PROTEIN AND SATELLITE RNAIN INSECT CELLS AND 3-DIMENSIONAL STRUCTURE OF TOBACCO RINGSPOT VIRUS-LIKE PARTICLES
S. Singh et al., EXPRESSION OF TOBACCO RINGSPOT VIRUS CAPSID PROTEIN AND SATELLITE RNAIN INSECT CELLS AND 3-DIMENSIONAL STRUCTURE OF TOBACCO RINGSPOT VIRUS-LIKE PARTICLES, Virology, 213(2), 1995, pp. 472-481
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.