Reovirus Virion-Like Particles Obtained by Recoating Infectious Subvirion Particles with Baculovirus-Expressed ς3 Protein: an Approach for Analyzing ς3 Functions during Virus Entry

Abstract

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein ς3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed ς3 protein to infectious subvirion particles (ISVPs) that lack ς3. A level of ς3 binding approaching 100% of that in native virions was routinely achieved. The ς3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study ς3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of ς3 but contain outer-capsid protein μ1/μ1C mostly cleaved at the δ-φ junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that ς3, and not the δ-φ cleavage of μ1/μ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric ς3 proteins to localize the primary determinants of a strain-dependent difference in ς3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

Selection and characterization of a reovirus mutant with improved thermostability

“…To determine if HR mutations alter disassembly kinetics, virions were digested in vitro with endoproteinase LysC (EKC). EKC probes for subtle differences in structure (57, 59). T1L and T1L μ1 M258I σ3 were degraded within 40 min.…”

The reovirus μ1 protein contributes to the environmental stability of virions

“…During cell entry, virions are proteolytically disassembled to generate a metastable intermediate, called infectious subviral particle (ISVP) (17, 26–33). The viral and environmental factors that influence ISVP stability have been thoroughly investigated (38, 41, 47–52, 54–67). Notably, heat induces loss of infectivity, which is accompanied by a conformational change within the μ1 outer capsid protein (48).…”

“…Notably, heat induces loss of infectivity, which is accompanied by a conformational change within the μ1 outer capsid protein (48). Virions are more resistant to thermal inactivation than ISVPs; the σ3 outer capsid protein confers stability by capping μ1 (41, 48, 52, 54). Nonetheless, whether μ1-σ3 interactions prevent the virion from adopting an altered conformation is undetermined.…”

“…Proteolytic disassembly of the reovirus outer capsid (i.e., virion-to-ISVP conversion) is regulated by a subset of σ3 residues; subtle changes in structure are correlated with altered protease sensitivity (53, 54, 68–70). Mismatched (or imperfect) outer capsid interactions could produce such a change, which may not be discernable at the structural resolutions determined above (Fig.…”

“…When comparing reovirus strains (e.g., type 1 Lang [T1L] and type 3 Dearing [T3D]), differences in the efficiency of inactivation (inversely related to stability) map to either the M2 gene segment (encodes for μ1) or the S4 gene segment (encodes for σ3) (9). Furthermore, virions are more resistant to thermal inactivation than ISVPs; σ3 confers stability by capping μ1 (41, 48, 52, 54). Mutations in σ3 are correlated with altered sensitivity to heating (53).…”