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Title
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Expression of tobacco ringspot virus capsid protein and satellite RNA in insect cells and three-dimensional structure of tobacco ringspot virus-like particles.
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Authors
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S.Singh,
R.Rothnagel,
B.V.Prasad,
B.Buckley.
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Ref.
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Virology, 1995,
213,
472-481.
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PubMed id
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Abstract
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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 generated in 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-A
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.
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