title: Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry.
abstract: Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.
authors.affiliation: Department of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Zhaozhu
authors.initials: Z
authors.lastname: Qiu
authors.forename: Susan T
authors.initials: ST
authors.lastname: Hingley
authors.forename: Graham
authors.initials: G
authors.lastname: Simmons
authors.forename: Christopher
authors.initials: C
authors.lastname: Yu
authors.forename: Jayasri
authors.initials: J
authors.lastname: Das Sarma
authors.forename: Paul
authors.initials: P
authors.lastname: Bates
authors.forename: Susan R
authors.initials: SR
authors.lastname: Weiss
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D002459:Cell Fusion; D002460:Cell Line; D018352:Coronavirus Infections / Q000209:etiology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology*; D005347:Fibroblasts / Q000821:virology; D006023:Glycoproteins / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D006517:Murine hepatitis virus / Q000472:pathogenicity*; D014764:Viral Proteins / Q000502:physiology*
chemical_list: D006023:Glycoproteins; D014764:Viral Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534278:Ctsl protein, mouse