Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein

Mammalian reoviruses, prototype members of the Reoviridae family of nonenve loped double-stranded RNA viruses, use at least three proteins-sigma1, mu1, and sigma3-to enter host cells, sigma1, a major determinant of cell tropis m, mediates viral attachment to cellular receptors. Studies of sigma1 funct ions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral protei ns. To mitigate this problem, we produced virion-like particles by "recoati ng" genome-containing core particles that lacked sigma1, mu1, and sigma3 wi th recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they clos ely resembled native virions in three-dimensional structure, including feat ures attributable to sigma1. The recoated particles bound to and infected c ultured cells in a sigma1-dependent manner and were approximately 1 million times as infections as cores and 0.5 times as infectious as native virions . Experiments with recoated particles containing recombinant sigma1 from ei ther of two different reovirus strains confirmed that differences in cell a ttachment and infectivity previously observed between those strains are det ermined by the sigma1 protein. Additional experiments showed that recoated particles containing al proteins with engineered mutations can be used to a nalyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular g enetic dissections of sigma1 with respect to its structure, assembly into p articles, and roles in entry.