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* Laboratory of Structural Biology, The genomes of double-stranded (ds)RNA
viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment
Computational
Bioscience and Engineering Laboratory, Department of Biology, Brookhaven National Laboratory, Upton, New York 11973
the viral capsid. We have used
cryoelectron microscopy and three-dimensional image
reconstruction to study this compartment in the case of
L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution,
we distinguish multiple domains in the elongated Gag
subunits, whose nonequivalent packing is reflected in
subtly different morphologies of the two protomers.
Small holes, 10-15 Å across, perforate the capsid wall,
which functions as a molecular sieve, allowing the exit
of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes.
Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is
an A-form duplex, and that RNA filaments emanating
from disrupted virions often consist of two or more
closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered
inside full capsids, but it is packed relatively loosely; in
L-A, the center-to-center spacing between duplexes is
40-45 Å, compared with 25-30 Å in other double-stranded viruses. The looser packing of L-A RNA
allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the
polymerase immobilized on the inner capsid wall.
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