title: Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses.
abstract: RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.
authors.affiliation: Unité de Génomique Virale et Vaccination, Institut Pasteur, CNRS URA 3015, Paris, France.
authors.forename: Anastassia V
authors.initials: AV
authors.lastname: Komarova
authors.forename: Chantal
authors.initials: C
authors.lastname: Combredet
authors.forename: Laurène
authors.initials: L
authors.lastname: Meyniel-Schicklin
authors.forename: Manuel
authors.initials: M
authors.lastname: Chapelle
authors.forename: Grégory
authors.initials: G
authors.lastname: Caignard
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Camadro
authors.forename: Vincent
authors.initials: V
authors.lastname: Lotteau
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Vidalain
authors.forename: Frédéric
authors.initials: F
authors.lastname: Tangy
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D053487:DEAD-box RNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D057809:HEK293 Cells; D006655:Histone Deacetylases / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D000072640:Interferon-Induced Helicase, IFIH1; D008457:Measles / Q000378:metabolism / Q000821:virology*; D008459:Measles virus / Q000235:genetics / Q000502:physiology*; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D030781:Organisms, Genetically Modified; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D059386:Reverse Genetics; D050794:STAT1 Transcription Factor / Q000302:isolation & purification / Q000378:metabolism; D050795:STAT2 Transcription Factor / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D014709:Vero Cells; D037521:Virulence Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014779:Virus Replication
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; C494084:STAT2 protein, human; D037521:Virulence Factors; D006655:Histone Deacetylases; C452815:IFIH1 protein, human; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1