In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3.

Abstract

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.