Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection
2011; Elsevier BV; Volume: 10; Issue: 10 Linguagem: Inglês
10.1074/mcp.m111.009936
ISSN1535-9484
AutoresAiping Zhang, Chad D. Williamson, Daniel S. Wong, Matthew D. Bullough, Kristy J. Brown, Yetrib Hathout, Anamaris M. Colberg‐Poley,
Tópico(s)Metabolism and Genetic Disorders
ResumoEndoplasmic 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. 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. Congenital human cytomegalovirus (HCMV) 1The abbreviations used are:ANTadenine nucleotide translocatorArgArginineBADbacterial artificial chromosome of human cytomegalovirus strain AD169Ca2+calciumDMEMDulbecco's Modified Eagle MediumERendoplasmic reticulumETCelectron transport chainGRPglucose regulated proteinHCMVhuman cytomegalovirusHFFshuman foreskin fibroblastshpihours post infectionHSPheat shock proteinIP3Rinositol 1,4,5-triphosphate receptorLTQlinear trap quadrupoleLysLysineMAMmitochondria-associated membranesMfnmitofusinminminutesOMMouter mitochondrial membranePBSphosphate buffered salinePGMphosphoglycerate mutasepUL37x1UL37 exon 1 proteinSig-1Rsigma 1 receptorSILACstable isotope labeling by amino acids in cell cultureSRsignal recognition particle receptorSRPsignal recognition particleTCAtricarboxylic acidVDACvoltage dependent anion channelvMIAviral mitochondria localized inhibitor of apoptosiswtwild type. infection is the leading viral cause of birth defects, including sensorineuronal hearing loss and developmental delay, in developed countries (1Mocarski E.S. Shenk T. Pass R.F. Cytomegaloviruses.in: Knipe D.M. Howley P.M. Fields Virology. 5th Ed. Wolters Kluwer Health, Lippincott Williams & Wilkins, Philadelphia2007: 2701-2772Google Scholar, 2Gerna G. Baldanti F. Revello M.G. Pathogenesis of human cytomegalovirus infection and cellular targets.Hum. Immunol. 2004; 65: 381-386Crossref PubMed Scopus (101) Google Scholar, 3Boeckh M. Nichols W.G. The impact of cytomegalovirus serostatus of donor and recipient before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive therapy.Blood. 2004; 103: 2003-2008Crossref PubMed Scopus (307) Google Scholar). Most HCMV infections in immunocompetent persons are asymptomatic. Nonetheless, HCMV infection is a major cause of morbidity and mortality in immunocompromised individuals (1Mocarski E.S. Shenk T. Pass R.F. Cytomegaloviruses.in: Knipe D.M. Howley P.M. Fields Virology. 5th Ed. Wolters Kluwer Health, Lippincott Williams & Wilkins, Philadelphia2007: 2701-2772Google Scholar, 2Gerna G. Baldanti F. Revello M.G. Pathogenesis of human cytomegalovirus infection and cellular targets.Hum. Immunol. 2004; 65: 381-386Crossref PubMed Scopus (101) Google Scholar, 3Boeckh M. Nichols W.G. The impact of cytomegalovirus serostatus of donor and recipient before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive therapy.Blood. 2004; 103: 2003-2008Crossref PubMed Scopus (307) Google Scholar). Severe manifestations of HCMV infection in these patients include interstitial pneumonia, hepatitis, meningoencephalitis, gastrointestinal disease, myocarditis, bone marrow suppression, and retinitis. Because of its impact on human suffering and the health care system, the Institute of Medicine has assigned the highest priority to HCMV vaccine development (4Khanna R. Diamond D.J. Human cytomegalovirus vaccine: time to look for alternative options.Trends Mol. Med. 2006; 12: 26-33Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). adenine nucleotide translocator Arginine bacterial artificial chromosome of human cytomegalovirus strain AD169 calcium Dulbecco's Modified Eagle Medium endoplasmic reticulum electron transport chain glucose regulated protein human cytomegalovirus human foreskin fibroblasts hours post infection heat shock protein inositol 1,4,5-triphosphate receptor linear trap quadrupole Lysine mitochondria-associated membranes mitofusin minutes outer mitochondrial membrane phosphate buffered saline phosphoglycerate mutase UL37 exon 1 protein sigma 1 receptor stable isotope labeling by amino acids in cell culture signal recognition particle receptor signal recognition particle tricarboxylic acid voltage dependent anion channel viral mitochondria localized inhibitor of apoptosis wild type. Like that of most viruses, HCMV growth in permissive cells requires a large supply of energy and substrates from the infected cell (5Munger J. Bajad S.U. Coller H.A. Shenk T. Rabinowitz J.D. Dynamics of the cellular metabolome during human cytomegalovirus infection.PLoS Pathog. 2006; 2: e132Crossref PubMed Scopus (309) Google Scholar, 6Munger J. Bennett B.D. Parikh A. Feng X.J. McArdle J. Rabitz H.A. Shenk T. Rabinowitz J.D. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (495) Google Scholar, 7Chambers J.W. Maguire T.G. Alwine J.C. Glutamine metabolism is essential for human cytomegalovirus infection.J. Virol. 2010; 84: 1867-1873Crossref PubMed Scopus (164) Google Scholar). To obtain these, HCMV infection causes a global metabolic up-regulation of central carbon metabolic flux (5Munger J. Bajad S.U. Coller H.A. Shenk T. Rabinowitz J.D. Dynamics of the cellular metabolome during human cytomegalovirus infection.PLoS Pathog. 2006; 2: e132Crossref PubMed Scopus (309) Google Scholar, 6Munger J. Bennett B.D. Parikh A. Feng X.J. McArdle J. Rabitz H.A. Shenk T. Rabinowitz J.D. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (495) Google Scholar). In particular, HCMV infection induces increased glucose and glutamine consumption during permissive infection of human foreskin fibroblasts (HFFs) (5Munger J. Bajad S.U. Coller H.A. Shenk T. Rabinowitz J.D. Dynamics of the cellular metabolome during human cytomegalovirus infection.PLoS Pathog. 2006; 2: e132Crossref PubMed Scopus (309) Google Scholar, 6Munger J. Bennett B.D. Parikh A. Feng X.J. McArdle J. Rabitz H.A. Shenk T. Rabinowitz J.D. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (495) Google Scholar, 7Chambers J.W. Maguire T.G. Alwine J.C. Glutamine metabolism is essential for human cytomegalovirus infection.J. Virol. 2010; 84: 1867-1873Crossref PubMed Scopus (164) Google Scholar). Increased aerobic glycolysis allows HCMV to use glucose biosynthetically, wherein most of the acetyl coenzyme A supports fatty acid synthesis needed for membrane formation for its progeny viruses. Thus, HCMV growth reprograms cellular mitochondrial metabolic flux and institutes its own metabolic program. Because of its protracted growth cycle over several days, productive HCMV infection further requires antiapoptotic activity for efficient progeny production (8Reboredo M. Greaves R.F. Hahn G. Human cytomegalovirus proteins encoded by UL37 exon 1 protect infected fibroblasts against virus-induced apoptosis and are required for efficient virus replication.J. Gen. Virol. 2004; 85: 3555-3567Crossref PubMed Scopus (78) Google Scholar). To extend cell viability, HCMV encodes several products to undermine mitochondrial-mediated apoptosis. The HCMV UL36–38 locus encodes five proteins with antiapoptotic activities, including the predominant UL37 exon 1 protein (pUL37×1), also known as viral mitochondria localized inhibitor of apoptosis (vMIA), the lower abundance UL37Medium protein and the UL37 glycoprotein, the UL36 protein and the early UL38 protein (9Kouzarides T. Bankier A.T. Satchwell S.C. Preddy E. Barrell B.G. An immediate early gene of human cytomegalovirus encodes a potential membrane glycoprotein.Virology. 1988; 165: 151-164Crossref PubMed Scopus (84) Google Scholar, 10Goldmacher V.S. Bartle L.M. Skaletskaya A. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. Cahir McFarland E.D. Kieff E.D. Mocarski E.S. Chittenden T. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12536-12541Crossref PubMed Scopus (365) Google Scholar, 11Terhune S. Torigoi E. Moorman N. Silva M. Qian Z. Shenk T. Yu D. Human cytomegalovirus UL38 protein blocks apoptosis.J. Virol. 2007; 81: 3109-3123Crossref PubMed Scopus (122) Google Scholar, 12McCormick A.L. Roback L. Livingston-Rosanoff D. St Clair C. The human cytomegalovirus UL36 gene controls caspase-dependent and -independent cell death programs activated by infection of monocytes differentiating to macrophages.J. Virol. 2010; 84: 5108-5123Crossref PubMed Scopus (51) Google Scholar). pUL37×1 blocks mitochondrial-mediated apoptosis by translocating proapoptotic Bax to the outer mitochondrial membrane (OMM) and suppressing its proapoptotic activity (13Arnoult D. Bartle L.M. Skaletskaya A. Poncet D. Zamzami N. Park P.U. Sharpe J. Youle R.J. Goldmacher V.S. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7988-7993Crossref PubMed Scopus (173) Google Scholar, 14Poncet D. Larochette N. Pauleau A.L. Boya P. Jalil A.A. Cartron P.F. Vallette F. Schnebelen C. Bartle L.M. Skaletskaya A. Boutolleau D. Martinou J.C. Goldmacher V.S. Kroemer G. Zamzami N. An anti-apoptotic viral protein that recruits Bax to mitochondria.J. Biol. Chem. 2004; 279: 22605-22614Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15McCormick A.L. Meiering C.D. Smith G.B. Mocarski E.S. Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis.J. Virol. 2005; 79: 12205-12217Crossref PubMed Scopus (66) Google Scholar). In addition, pUL37×1/vMIA has been reported to alter mitochondrial ATP synthesis by decreasing the activity of the mitochondrial phosphate carrier in transfected cells (16Poncet D. Pauleau A.L. Szabadkai G. Vozza A. Scholz S.R. Le Bras M. Brière J.J. Jalil A. Le Moigne R. Brenner C. Hahn G. Wittig I. Schägger H. Lemaire C. Bianchi K. Souquere S. Pierron G. Rustin P. Goldmacher V.S. Rizzuto R. Palmieri F. Kroemer G. Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA.J. Cell Biol. 2006; 174: 985-996Crossref PubMed Scopus (81) Google Scholar). By early times, HCMV infection also encodes an untranslated beta 2.7 RNA that localizes to mitochondria (17Spector D.H. Activation and regulation of human cytomegalovirus early genes.Intervirology. 1996; 39: 361-377Crossref PubMed Scopus (61) Google Scholar, 18Greenaway P.J. Wilkinson G.W. Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169.Virus Res. 1987; 7: 17-31Crossref PubMed Scopus (44) Google Scholar). Beta 2.7 RNA interacts with the GRIM-19 subunit of mitochondrial complex I and prevents its re-localization from the inner mitochondrial membrane to discrete perinuclear sites (19Reeves M.B. Davies A.A. McSharry B.P. Wilkinson G.W. Sinclair J.H. Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death.Science. 2007; 316: 1345-1348Crossref PubMed Scopus (210) Google Scholar). Thus, HCMV infection redundantly blocks mitochondrial-mediated proapoptotic signaling pathways. Mitochondrial activities, including its metabolic pathways and induction of apoptosis, are modulated by contacts with endoplasmic reticulum (ER) subdomains, known as mitochondria-associated membranes (MAM) (20Hayashi T. Rizzuto R. Hajnoczky G. Su T.P. MAM: more than just a housekeeper.Trends Cell Biol. 2009; 19: 81-88Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 21Cárdenas C. Miller R.A. Smith I. Bui T. Molgó J. Müller M. Vais H. Cheung K.H. Yang J. Parker I. Thompson C.B. Birnbaum M.J. Hallows K.R. Foskett J.K. Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.Cell. 2010; 142: 270-283Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). Direct ER-mitochondrial contacts have been best visualized by electron tomography (22Csordás G. Renken C. Várnai P. Walter L. Weaver D. Buttle K.F. Balla T. Mannella C.A. Hajnóczky G. Structural and functional features and significance of the physical linkage between ER and mitochondria.J. Cell Biol. 2006; 174: 915-921Crossref PubMed Scopus (967) Google Scholar). The physical distance between the ER membrane and OMM is ∼10 nm. The distance between the organelles is larger (∼25 nm) when ribosomes are found between the juxtaposed membranes. Protein tethers can be seen between the ER and OMM. Mitofusin 2 (Mfn2) (23de Brito O.M. Scorrano L. Mitofusin 2 tethers endoplasmic reticulum to mitochondria.Nature. 2008; 456: 605-610Crossref PubMed Scopus (1718) Google Scholar, 24de Brito O.M. Scorrano L. Mitofusin-2 regulates mitochondrial and endoplasmic reticulum morphology and tethering: the role of Ras.Mitochondrion. 2009; 9: 222-226Crossref PubMed Scopus (115) Google Scholar), mitostatin (25Cerqua C. Anesti V. Pyakurel A. Liu D. Naon D. Wiche G. Baffa R. Dimmer K.S. Scorrano L. Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition.EMBO Rep. 2010; 11: 854-860Crossref PubMed Scopus (102) Google Scholar), PACS-2 (26Simmen T. Aslan J.E. Blagoveshchenskaya A.D. Thomas L. Wan L. Xiang Y. Feliciangeli S.F. Hung C.H. Crump C.M. Thomas G. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis.EMBO J. 2005; 24: 717-729Crossref PubMed Scopus (420) Google Scholar), and a calcium (Ca2+) signaling complex (27Szabadkai G. Bianchi K. Várnai P. De Stefani D. Wieckowski M.R. Cavagna D. Nagy A.I. Balla T. Rizzuto R. Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels.J. Cell Biol. 2006; 175: 901-911Crossref PubMed Scopus (932) Google Scholar), have been implicated in stabilizing contacts between the ER and the OMM. Our previous work demonstrated that HCMV infection targets ER-mitochondrial contacts by trafficking of its pUL37×1 and the N-terminal UL37 glycoprotein fragment (28Bozidis P. Williamson C.D. Colberg-Poley A.M. Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells.J. Virol. 2008; 82: 2715-2726Crossref PubMed Scopus (34) Google Scholar, 29Bozidis P. Williamson C.D. Wong D.S. Colberg-Poley A.M. Trafficking of UL37 proteins into mitochondrion-associated membranes during permissive human cytomegalovirus infection.J. Virol. 2010; 84: 7898-7903Crossref PubMed Scopus (35) Google Scholar, 30Williamson C.D. Colberg-Poley A.M. Access of viral proteins to mitochondria via mitochondria-associated membranes.Rev. Med. Virol. 2009; 19: 147-164Crossref PubMed Scopus (53) Google Scholar, 31Williamson C.D. Colberg-Poley A.M. Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria.J. Virol. 2010; 84: 6400-6409Crossref PubMed Scopus (19) Google Scholar, 32Williamson C.D. Zhang A. Colberg-Poley A.M. The human cytomegalovirus UL37 exon 1 protein associates with internal lipid rafts.J. Virol. 2011; 85: 2100-2111Crossref PubMed Scopus (0) Google Scholar, 33Mavinakere M.S. Colberg-Poley A.M. Internal cleavage of the human cytomegalovirus UL37 immediate-early glycoprotein and divergent trafficking of its proteolytic fragments.J. Gen. Virol. 2004; 85: 1989-1994Crossref PubMed Scopus (13) Google Scholar, 34Mavinakere M.S. Colberg-Poley A.M. Dual targeting of the human cytomegalovirus UL37 exon 1 protein during permissive infection.J. Gen. Virol. 2004; 85: 323-329Crossref PubMed Scopus (32) Google Scholar). A highly conserved UL37×1 leader drives ER translocation, MAM association, and subsequent trafficking to the OMM (31Williamson C.D. Colberg-Poley A.M. Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria.J. Virol. 2010; 84: 6400-6409Crossref PubMed Scopus (19) Google Scholar, 34Mavinakere M.S. Colberg-Poley A.M. Dual targeting of the human cytomegalovirus UL37 exon 1 protein during permissive infection.J. Gen. Virol. 2004; 85: 323-329Crossref PubMed Scopus (32) Google Scholar). pUL37×1 traffics from the ER, where it causes Ca2+ efflux (35Sharon-Friling R. Goodhouse J. Colberg-Poley A.M. Shenk T. Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19117-19122Crossref PubMed Scopus (106) Google Scholar), associates with internal MAM lipid rafts (36Williamson C.D. Zhang A. Colberg-Poley A.M. The human cytomegalovirus protein UL37 exon 1 associates with internal lipid rafts.J. Virol. 2011; 85: 2100-2111Crossref PubMed Scopus (33) Google Scholar) in close proximity to sigma 1 receptor (Sig-1R) (32Williamson C.D. Zhang A. Colberg-Poley A.M. The human cytomegalovirus UL37 exon 1 protein associates with internal lipid rafts.J. Virol. 2011; 85: 2100-2111Crossref PubMed Scopus (0) Google Scholar) and translocates to the OMM where it has potent antiapoptotic activity by recruiting Bax to the OMM and inhibiting its proapoptotic activities (10Goldmacher V.S. Bartle L.M. Skaletskaya A. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. Cahir McFarland E.D. Kieff E.D. Mocarski E.S. Chittenden T. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12536-12541Crossref PubMed Scopus (365) Google Scholar, 13Arnoult D. Bartle L.M. Skaletskaya A. Poncet D. Zamzami N. Park P.U. Sharpe J. Youle R.J. Goldmacher V.S. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7988-7993Crossref PubMed Scopus (173) Google Scholar, 14Poncet D. Larochette N. Pauleau A.L. Boya P. Jalil A.A. Cartron P.F. Vallette F. Schnebelen C. Bartle L.M. Skaletskaya A. Boutolleau D. Martinou J.C. Goldmacher V.S. Kroemer G. Zamzami N. An anti-apoptotic viral protein that recruits Bax to mitochondria.J. Biol. Chem. 2004; 279: 22605-22614Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 37Pauleau A.L. Larochette N. Giordanetto F. Scholz S.R. Poncet D. Zamzami N. Golmacher V.S. Kroemer G. Structure-function analysis of the interaction between Bax and the cytomegalovirus-encoded protein vMIA.Oncogene. 2007; 26: 7067-7080Crossref PubMed Scopus (45) Google Scholar, 38Norris K.L. Youle R.J. Cytomegalovirus proteins vMIA and m38.5 link mitochondrial morphogenesis to Bcl-2 family proteins.J. Virol. 2008; 82: 6232-6243Crossref PubMed Scopus (63) Google Scholar). In addition to targeting the MAM by pUL37×1, we found that HCMV infection increases the abundance of cytosolic glucose regulated protein 75 (GRP75), a component of the MAM Ca2+ macromolecular complex (29Bozidis P. Williamson C.D. Wong D.S. Colberg-Poley A.M. Trafficking of UL37 proteins into mitochondrion-associated membranes during permissive human cytomegalovirus infection.J. Virol. 2010; 84: 7898-7903Crossref PubMed Scopus (35) Google Scholar). Thus, HCMV positions itself to control ER-mitochondrial cross-talk and thereby modulate key mitochondrial functions. By targeting the MAM, HCMV predictably acquires another level of control over mitochondrial metabolism and mitochondrial-mediated apoptosis. To gain insight into how HCMV infection alters the functionality of ER-mitochondrial contacts to its advantage, we undertook quantitative analyses of the MAM proteome using stable isotope labeling by amino acids in cell culture (SILAC) (39Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar). A late time of infection was chosen for these studies as HCMV infection dramatically alters metabolic flux (5Munger J. Bajad S.U. Coller H.A. Shenk T. Rabinowitz J.D. Dynamics of the cellular metabolome during human cytomegalovirus infection.PLoS Pathog. 2006; 2: e132Crossref PubMed Scopus (309) Google Scholar, 6Munger J. Bennett B.D. Parikh A. Feng X.J. McArdle J. Rabitz H.A. Shenk T. Rabinowitz J.D. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (495) Google Scholar, 7Chambers J.W. Maguire T.G. Alwine J.C. Glutamine metabolism is essential for human cytomegalovirus infection.J. Virol. 2010; 84: 1867-1873Crossref PubMed Scopus (164) Google Scholar), the cell secretome (40Dumortier J. Streblow D.N. Moses A.V. Jacobs J.M. Kreklywich C.N. Camp D. Smith R.D. Orloff S.L. Nelson J.A. Human cytomegalovirus secretome contains factors that induce angiogenesis and wound healing.J. Virol. 2008; 82: 6524-6535Crossref PubMed Scopus (96) Google Scholar), widespread changes in ER Ca2+ efflux (35Sharon-Friling R. Goodhouse J. Colberg-Poley A.M. Shenk T. Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19117-19122Crossref PubMed Scopus (106) Google Scholar), and a mitigated ER stress response (41Isler J.A. Skalet A.H. Alwine J.C. Human cytomegalovirus infection activates and regulates the unfolded protein response.J. Virol. 2005; 79: 6890-6899Crossref PubMed Scopus (231) Google Scholar) at these times; all events which are likely mediated by the MAM or to affect MAM domains. To date, no quantitative or nonquantitative proteomic analysis of the MAM fraction has yet been reported. Although proteomic analyses have identified HCMV virion components (42Varnum S.M. Streblow D.N. Monroe M.E. Smith P. Auberry K.J. Pasa-Tolic L. Wang D. Camp 2nd, D.G. Rodland K. Wiley S. Britt W. Shenk T. Smith R.D. Nelson J.A. Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.J. Virol. 2004; 78: 10960-10966Crossref PubMed Scopus (468) Google Scholar) and cellular proteins that interact with HCMV products (43Moorman N.J. Sharon-Friling R. Shenk T. Cristea I.M. A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation.Mol. Cell. Proteomics. 2010; 9: 851-860Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 44Gao Y. Colletti K. Pari G.S. Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis.J. Virol. 2008; 82: 96-104Crossref PubMed Scopus (53) Google Scholar) as well as defined the HCMV-induced secretome (40Dumortier J. Streblow D.N. Moses A.V. Jacobs J.M. Kreklywich C.N. Camp D. Smith R.D. Orloff S.L. Nelson J.A. Human cytomegalovirus secretome contains factors that induce angiogenesis and wound healing.J. Virol. 2008; 82: 6524-6535Crossref PubMed Scopus (96) Google Scholar), this is the first quantitative proteomic analyses of HCMV-infected HFFs and the effects of virus on ER-mitochondrial contacts. Using this quantitative proteomic approach, we observed dramatic changes in the MAM proteome by late times of HCMV infection, consistent with its global reprogramming of cellular metabolism. The association of chaperones, translation and ER-translocation machineries, Ca2+ signaling complexes, metabolic enzymes, and apoptotic regulatory proteins with the MAM fraction increased by late times of infection. These results suggest that HCMV infection induces a restructuring of ER-mitochondrial contacts by late times. This restructuring will predictably result in increased protein synthesis at the MAM, cell survival and bioenergetics. Further the increased abundance of mitochondrial inner mitochondrial membrane, intermembrane space and matrix proteins in the MAM fraction suggest that HCMV infection increases the stability of ER-mitochondrial contacts by late times of infection. HFFs were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum (FCS), 100 U/ml of penicillin, 100 μg/ml of streptomycin (Invitrogen) as described (45Adair R. Liebisch G.W. Su Y. Colberg-Poley A.M. Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection.J. Gen. Virol. 2004; 85: 3541-3553Crossref PubMed Scopus (21) Google Scholar). The bacterial artificial chromosome of HCMV strain AD169 (BAD) wild type (wt) was grown and titered as previously described (35Sharon-Friling R. Goodhouse J. Colberg-Poley A.M. Shenk T. Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19117-1
Referência(s)