title: Identification of host factors involved in coronavirus replication by quantitative proteomics analysis.
abstract: In this study, we applied a quantitative proteomic approach, based on SILAC, to investigate the interactions of coronaviruses with the secretory pathway of the host cell, with the aim to identify host factors involved in coronavirus replication. Comparison of the protein profiles of Golgi-enriched fractions of cells that were either mock infected or infected with mouse hepatitis virus revealed the significant depletion or enrichment of 116 proteins. Although ribosomal/nucleic acid binding proteins were enriched in the Golgi-fractions of mouse hepatitis virus-infected cells, proteins annotated to localize to several organelles of the secretory pathway were overrepresented among the proteins that were depleted from these fractions upon infection. We hypothesized that proteins, of which the abundance or distribution is affected by infection, are likely to be involved in the virus life cycle. Indeed, depletion of a small subset of the affected proteins by using small interfering RNAs identified several host factors involved in coronavirus infection. Transfection of small interfering RNAs targeting either C11orf59 or Golgi apparatus glycoprotein 1 resulted in increased virus replication, whereas depletion of vesicle-trafficking protein vesicle-trafficking protein sec22b enhanced the release of infectious progeny virus. Overexpression of these proteins, on the other hand, had a negative effect on virus replication. Overall, our study shows that the SILAC approach is a suitable tool to study host-pathogen interactions and to identify host proteins involved in virus replication.
authors.affiliation: Department of Biochemistry and Cell Biology, Biochemistry Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Mijke W
authors.initials: MW
authors.lastname: Vogels
authors.forename: Bas W M
authors.initials: BW
authors.lastname: van Balkom
authors.forename: Dora V
authors.initials: DV
authors.lastname: Kaloyanova
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Batenburg
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Heck
authors.forename: J Bernd
authors.initials: JB
authors.lastname: Helms
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
mesh_terms: D017934:Coronavirus / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000502:physiology*; D006801:Humans; D013058:Mass Spectrometry; D006517:Murine hepatitis virus / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; D017468:Receptors, Fibroblast Growth Factor; D012795:Sialoglycoproteins; C086711:cysteine-rich fibroblast growth factor receptor