Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry.

Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.

Immunoelectron microscopic demonstration of regulated pathway for calcitonin and constitutive pathway for carcinoembryonic antigen in the same cells of human medullary carcinomas of thyroid glands.

The human medullary carcinomas are well known to secrete calcitonin (CT) as a neuroendocrine peptide and carcinoembryonic antigen (CEA) as a serum protein that is integrated into the cell membrane. This ultrastructural study is designed to elucidate whether CT and CEA are secreted via two different intracellular secretory pathways, a regulated pathway and a constitutive pathway. The immunoelectron microscopic postembedding method, performed on four cases of the human medullary carcinomas of the thyroid using plastic embedding material, disclosed two distinct different localization patterns for CT and CEA. CT was localized exclusively in dense cored secretory granules. CEA was present in the cell membrane and in the secretory vesicles. The secretory granules were completely negative for CEA. The trans-Golgi networks were also positive for CT and CEA. Electron microscopic double staining confirmed these localization in the same carcinoma cells. These observations suggest the presence of two distinct pathways in the endocrine cancer cells, i.e., the regulated pathway for CT and the constitutive pathway for CEA.