title: Evaluating Exosome Protein Content Changes Induced by Virus Activity Using SILAC Labeling and LC-MS/MS.
abstract: Exosomes are small membrane vesicles that are produced by cells and excreted into extracellular space. Contents of exosomes generally include lipid, membrane, and soluble proteins, and various types of coding and noncoding RNAs. Over the past decades, it has become clear that exosomes constitute an important vector for intercellular transport and communication with significant functional relevance. Evaluating exosome contents and their changes are vital for understanding its role in different physiological and pathological processes. Infection by certain pathogens, including viruses as well as intracellular bacteria, fungi, and parasites, has been shown to induce specific content changes in exosomes produced by infected cells. Evidences also indicate that exosomes produced by infected cells may actively participate in host-virus interactions, including immune responses. Studies of exosome content changes involve highly complex experimental and computational procedures, which can become even more complicated in the context of viral infections, due to the production and secretion of multiple virus-derived proteins and particles by infected cells. In this chapter, general and specific considerations relating to studies of exosome content changes induced by virus activities are discussed and illustrated with the detailed protocols previously used to identify protein content changes in Huh-7 cell exosomes induced by transfection with hepatitis B virus replicon plasmids, using SILAC labeling and LS-MS/MS. Hopefully, this would help enable more and further studies along similar lines and enhance the understanding of this new aspect of host-pathogen interactions.
authors.affiliation: Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, PR China; Microbiology Laboratory, Shanghai Municipal Center for Disease Control and Prevention, Shanghai, PR China.
authors.forename: X
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, PR China.
authors.forename: Y
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, PR China. Electronic address: liujing212@fudan.edu.cn.
authors.forename: J
authors.initials: J
authors.lastname: Liu
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D055354:Exosomes / Q000737:chemistry* / Q000378:metabolism / Q000821:virology; D006515:Hepatitis B virus / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D007553:Isotope Labeling; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome
keywords: Exosome; LC-MS/MS; Proteomic analysis; SILAC; Virion morphogenesis; Virus