In Vitro Recoating of Reovirus Cores with Baculovirus-Expressed Outer-Capsid Proteins μ1 and ς3

Abstract

Reovirus outer-capsid proteins μ1, ς3, and ς1 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 μ1 and ς3 onto purified cores that lack μ1, ς3, and ς1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ς1), 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 μ1 and ς3 onto cores had induced rearrangement of the pentameric λ2 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 ς1 protein. To examine the utility of r-cores for genetic dissections of μ1 functions in reovirus entry, we generated r-cores containing a mutant form of μ1 that had been engineered to resist cleavage at the δ:φ junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in δ:φ 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.

Peptides released from reovirus outer capsid form membrane pores that recruit virus particles

“…In contrast to prior evidence for μ1N, protease‐mediated C‐terminal cleavage of μ1, generating the C‐terminal 13‐kDa fragment ϕ (Nibert and Fields, 1992), is not required for haemolysis or infection (Chandran and Nibert, 1998; Chandran et al , 1999). As the ϕ region remains tethered to particles in the absence of cleavage, its role in these activities has not been excluded.…”

“…These results confirm that at least a portion of ϕ is released from ISVP * s and implicate ϕ as another candidate in haemolysis activity. We do not expect σ1 to contribute, because previous RBC + reactions using recoated cores without σ1 showed normal levels of haemolysis (Chandran et al , 1999).…”

“…We transferred N42Aprc * spin‐reaction supernatants to tubes with either RBCs or doubly labelled 3K/10K or 10K/40K RGs (Figure 5A and B). We compared these supernatants with those generated from ISVP * ‐like particles derived from cores recoated with wild‐type μ1 and σ3 (WTprc * s) (Chandran et al , 1999) (Figure 5A and B). Both the N42Aprcs and the WTprcs were recoated without σ1, so both types of supernatant lacked σ1.…”

Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein μNS during reovirus infection

“…Purified virions, grown in L929 cells and purified as described elsewhere (Nibert and Fields, 1992), were used for all infections. Cores were prepared as previously described (Chandran et al, 1999).…”

Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

“…Primary antibodies used were as follows: monoclonal mouse anti-FLAG (α-FLAG) antibody (Sigma Aldrich, F1804), monoclonal mouse α-HA antibody (ThermoFisher, 26183), polyclonal mouse α-μNS, polyclonal rabbit α-μNS, α-μ2, and T1L α-virion antibodies (9, 27-29), monoclonal mouse α-σNS (3E10) and α-λ2 (7F4) antibodies deposited in the Developmental Studies Hybridoma Bank (DSHB) by Dermody, T.S. (30, 31), and rabbit α-MRV(core) (32). Secondary antibodies used are Alexa 594- and 488-conjugated donkey anti-mouse or anti-rabbit IgG antibodies (Invitrogen Life Technologies).…”