In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu 1 and sigma 3

Reovirus outer-capsid proteins mu 1, sigma 3, and sigma 1 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 mu 1 and sigma 3 onto purif ied cores that lack mu 1, sigma 3, and sigma 1. The resulting particles (re coated cores, or r-cores) closely resembled native virions in protein compo sition (except for lacking cell attachment protein sigma 1), buoyant densit y, and particle morphology by scanning cryoelectron microscopy. Transmissio n cryoelectron microscopy and image reconstruction of r-cores confirmed tha t they closely resembled virions in the structure of the outer capsid and r evealed that assembly of mu 1 and sigma 3 onto cores had induced rearrangem ent of the pentameric lambda 2 turrets into a conformation approximating th at in virions. r-cores, like virions, underwent proteolytic conversion to p articles resembling native ISVPs (infectious subvirion particles) in protei n composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murin e L, cells and, like virions but not ISVPs or cores, were inhibited from pr oductively infecting these cells by the presence of either NH4Cl or E-64. T he latter results suggest that r-cores and virions used similar routes of e ntry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma 1 protein. To examine the utility of r-cores for genetic dissections of mu 1 functions in reovirus e ntry, we generated r-cores containing a mutant form of mu 1 that had been e ngineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in del ta:phi cleavage, these ISVP-like particles were fully competent to permeabi lize membranes in vitro and to infect L cells in the presence of NH4Cl, pro viding new evidence that this cleavage is dispensable for productive infect ion.