An Unusual Aspartic Acid Cluster in the Reovirus Attachment Fiber σ1 Mediates Stability at Low pH and Preserves Trimeric Organization.

The reovirus attachment protein σ1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. σ1 is a trimeric filamentous protein with an α-helical coiled-coil tail, a triple-β-spiral body, and a globular head. At the trimer interface, the head domain features an unusual and conserved aspartic acid cluster, which forms the only significant intratrimer interactions in the head and must be protonated to allow trimer formation. To define the role of pH on σ1 stability and conformation, we tested its domains over a wide range of pH values. We show that all domains of σ1 are remarkably thermostable, even at the low pH of the stomach. We determined the optimal pH for stability to be between pHs 5 and 6, a value close to the pH of the endosome and of the jejunum. The σ1 head is stable at acidic and neutral pH but detrimerizes at basic pH. When Asp345 in the aspartic acid cluster is mutated to asparagine (D345N), the σ1 head loses stability at low pH and is more prone to detrimerize. Although the D345N mutation does not affect σ1 binding affinity for the JAM-A receptor, the overall binding stoichiometry is reduced by one-third. The additional replacement of the neighboring His349 with alanine disrupts inner trimer surface interactions, leading to a less thermostable and monomeric σ1 D345N head that fails to bind the JAM-A receptor. When the body is expressed together with the head domain, the thermostability is restored and the stoichiometry of the binding to JAM-A receptor is preserved. Our results confirm a fundamental role of the aspartic acid cluster as a pH-dependent molecular switch controlling trimerization and enhancing thermostability of σ1, which represent essential requirements to accomplish reovirus infection and entry and might be common mechanisms among other enteric viruses. IMPORTANCE Enteric viruses withstand the highly acidic environment of the stomach during transmission, and many of them use low pH as a trigger for conformational changes associated with entry. For many nonenveloped viruses, the structural basis of these effects is not clear. We have investigated the stability of the reovirus attachment protein σ1 over a range of pHs and find it to be remarkably thermostable, especially at low pH. We identify a role for the aspartic acid cluster in maintaining σ1 thermostability, trimeric organization, and binding to JAM-A receptor especially at the gastric pH reovirus has to withstand while passing the stomach. The understanding of monomer-trimer dynamics within σ1 enhances our knowledge of reovirus entry and has implications for stability and transmission of other enteric viruses.