title: Quantitative proteomic analyses of human cytomegalovirus-induced restructuring of endoplasmic reticulum-mitochondrial contacts at late times of infection.
abstract: Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.
authors: Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010, USA.
authors: Aiping
authors: A
authors: Zhang
authors: Chad D
authors: CD
authors: Williamson
authors: Daniel S
authors: DS
authors: Wong
authors: Matthew D
authors: MD
authors: Bullough
authors: Kristy J
authors: KJ
authors: Brown
authors: Yetrib
authors: Y
authors: Hathout
authors: Anamaris M
authors: AM
authors: Colberg-Poley
mesh_terms: D002853:Chromatography, Liquid; D003587:Cytomegalovirus; D003586:Cytomegalovirus Infections / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D007553:Isotope Labeling; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome