title: Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.
abstract: The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.
authors.affiliation: Department of Cell Biology, Utrecht University, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Klumperman
authors.forename: J K
authors.initials: JK
authors.lastname: Locker
authors.forename: A
authors.initials: A
authors.lastname: Meijer
authors.forename: M C
authors.initials: MC
authors.lastname: Horzinek
authors.forename: H J
authors.initials: HJ
authors.lastname: Geuze
authors.forename: P J
authors.initials: PJ
authors.lastname: Rottier
mesh_terms: D000818:Animals; D002415:Cats; D002460:Cell Line; D003332:Coronaviridae / Q000254:growth & development* / Q000378:metabolism / Q000648:ultrastructure; D016765:Coronavirus, Feline / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D005333:Fetus; D006056:Golgi Apparatus / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure; D006801:Humans; D001351:Infectious bronchitis virus / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D007668:Kidney; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D006517:Murine hepatitis virus / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D011995:Recombination, Genetic; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000032:analysis / Q000096:biosynthesis*
chemical_list: D014763:Viral Matrix Proteins