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© The Rockefeller University Press, 0021-9525/1997//975 $5.00
The Journal of Cell Biology, Volume 138, Number 5, , 1997 975-985

Article

Structure of L-A Virus: A Specialized Compartment for the Transcription and Replication of Double-stranded RNA



José R. Castón*, Benes L. Trus*,{ddagger}, Frank P. Booy*, Reed B. Wickner§, Joseph S. Wall||, and Alasdair C. Steven*

* Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases; {ddagger} Computational Bioscience and Engineering Laboratory, Division of Computer Research and Technology; § Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and || Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

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—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.

Abbreviations used in this paper: CTF, contrast transfer function; ds, double-stranded; ss, single-stranded; STEM, scanning transmission electron microscopy; TMV, tobacco mosaic virus.

Please address all correspondence to A.C. Steven, Building 6, Room B2-34, 6 Center Drive MSC 2717, National Institutes of Health, Bethesda MD 20892-2717. Tel.: (301) 496-0132. Fax: (301) 480-7629. e-mail: Alasdair_ Steven@NIH.GOV

J.R. Castón's present address is Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, 28049 Madrid, Spain.


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