title: Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent.
abstract: Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.
authors.affiliation: Department of Infectious Diseases and Immunology, Yalelaan 1, 3584CL Utrecht, The Netherlands.
authors.forename: Berend Jan
authors.initials: BJ
authors.lastname: Bosch
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011995:Recombination, Genetic; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D049452:Green Fluorescent Proteins