Abstract
Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.
Highlights
The reovirus outer capsid protein 3 acts as a substrate for intracellular proteases
To determine whether the Y354H polymorphism in type 3 Abney (T3A) 3 confers resistance to protease inhibitors, T3A, type 3 Dearing (T3D), and T3D-3Y354H were tested for the capacity to replicate in the presence of protease inhibitor E64
These findings suggest that one or more of the additional polymorphisms displayed by T3A 3 suppresses the Y354H phenotype and restores optimal 3 stability
Summary
The reovirus outer capsid protein 3 acts as a substrate for intracellular proteases. Variants with Y354H in 3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E These results identify a regulatory network of residues that control 3 cleavage and capsid stability, providing insight into the regulation of nonenveloped virus disassembly. 3 To whom correspondence should be addressed: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. The capsid proteins of nonenveloped viruses often contain hydrophobic motifs that are exposed in response to receptor binding or endosomal cues These motifs in turn disrupt host cell membranes and mediate viral access to the cytosol. Mutations that enhance outer capsid proteolysis, such as Y354H, accelerate the kinetics of viral entry into tissue culture cells and enable viral replication in settings of diminished protease availability This mutation is absent from most primary reovirus isolates [25], suggesting that it imposes a fitness penalty on the virus. We propose that residues 198 and 354 act in concert to maintain optimum stability of the reovirus capsid
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