The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability

The architecture of transmissible gastroenteritis coronavirus includes thre e different structural levels, the envelope, an internal core, and the nucl eocapsid that is released when the core is disrupted. Starting from purifie d virions, core structures have been reproducibly isolated as independent e ntities. The cores were stabilized at basic pH and by the presence of dival ent cations, with Mg2+ ions more effectively contributing to core stability . Core structures showed high resistance to different concentrations of det ergents, reducing agents, and urea and low concentrations of monovalent ion s (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domai n of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which result ed in destruction of the core structure. A specific ionic interaction betwe en the M protein carboxy terminus and the nucleocapsid was demonstrated usi ng three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and pur ified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein Co th e nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to t he indicated peptide but not by MAbs specific for the M protein amino termi nus; and (iii) a 26-residue peptide, including the predicted sequence (aa 2 37 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exp osed RNA by RNase treatment did not affect the binding. It is proposed that the hi protein is embedded within the virus membrane and that the C region , exposed to the interior face of the virion in a population of these molec ules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.