ABSTRACT: Reovirus particles are covered with 200 ?1/?3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of stepwise disassembly events. First, the ?3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the ?1 protein rearranges into a protease-sensitive conformation to generate ISVP*s and releases two virus-encoded peptides, ?1N and ?. The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by ?1 intratrimer, intertrimer, and trimer-core interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring ?1 trimers unwind and separate. Located within the ?1 jelly roll ? barrel domain, which is a known regulator of ISVP* formation, residues 340 to 343 form a loop and have been proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (?341 and ?342). Both deletions destabilized the outer capsid. Notably, ?342 impaired the viral life cycle; however, replicative fitness was restored by an additional change (V403A) within the ?1 jelly roll ? barrel domain. In the ?341 and ?342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also sufficiently flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.
