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

Chandran K, Zhang X, Olson NH, Walker SB, Chappell JD, Dermody TS, Baker TS, Nibert ML
J Virol. 2001 75 (11): 5335-42

PMID: 11333914 · PMCID: PMC114938 · DOI:10.1128/JVI.75.11.5335-5342.2001

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

MeSH Terms (15)

Baculoviridae Capsid Capsid Proteins Cell Line Cryoelectron Microscopy Eukaryotic Cells Hemagglutination Tests Hemagglutinins, Viral In Vitro Techniques Microscopy, Electron Recombinant Proteins Reoviridae Viral Proteins Virus Assembly Virus Replication

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