STRUCTURE OF L-A VIRUS - A SPECIALIZED COMPARTMENT FOR THE TRANSCRIPTION AND REPLICATION OF DOUBLE-STRANDED-RNA

The genomes of double-stranded (ds)RNA viruses are never exposed to th e cytoplasm but are confined to and replicated from a specialized prot ein-bound compartment-the viral capsid, We have used cryoelectron micr oscopy and three-dimensional image reconstruction to study this compar tment in the case of L-A, a yeast virus whose capsid consists of 60 as ymmetric dimers of Gag protein (76 kD). At 16-Angstrom resolution, we distinguish multiple domains in the elongated Gag subunits, whose none quivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 Angstrom across, perforate the caps id wall, which functions as a molecular sieve, allowing the exit of tr anscripts and the influx of metabolites, while retaining dsRNA and exc luding 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 protecti on 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 Angstrom, compared with 25 -30 Angstrom in other double-stranded viruses. The looser packing of L -A RNA allows for maneuverability in the crowded capsid interior, in w hich the genome (in both replication and transcription) must be transl ocated sequentially past the polymerase immobilized on the inner capsi d wall.