Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells

Bozidis, Petros; Williamson, Chad D.; Colberg-Poley, Anamaris M.

doi:10.1128/jvi.02456-07

Cited by 35 publications

(92 citation statements)

References 54 publications

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“…Each blot was stripped as previously described, and reprobed for the detection of other ER, MAM, mitochondrial markers (28,49). Blots were exposed to film (Denville Scientific, Metuchen, NJ) and the exposures were scanned using a Bio-Rad GS-800 Calibrated Densitometer and analyzed using QuantityOne Software.…”

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

“…Subcellular Fractionation and MAM Isolation-Each paired mixture was separately fractioned to isolate enriched heavy MAM from bulk ER and mitochondria as previously described (28,47). The Percoll method used for this analysis is one of the most rigorous procedures to obtain banded MAM and mitochondria.…”

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

“…Fixed cells were stored at 4°C until use. Cells were then probed with mouse anti-HSP60 IgG1 (Santa Cruz, 1:50), mouse anti-Hexokinase IgG1 (Santa Cruz, 1:50), or mouse anti-Sig-1R IgG1 (Santa Cruz; 1:50) and mouse anti-HCMV IE 1/2 IgG2A (Millipore, 1:50), and human anti-mitochondria IgG (Immunovision, 1:50) as described previously (28,49,50). Cells were then washed and probed simultaneously with 568 Alexa Fluor conjugated goat anti-mouse IgG1 (1:1500), 488 Alexa Fluor conjugated goat antimouse IgG2A (1:1500), and 647 Alexa Fluor conjugated goat antihuman IgG (1:1500) secondary antibodies.…”

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

“…Western Blot Analysis-Total or fractionated proteins were resolved by SDS-PAGE in 4 -12% Bis-Tris NuPage gels (Invitrogen) and analyzed by Western analyses as previously described (28,49). Blots were probed with the following primary antibodies: rabbit anti-UL37ϫ1 (amino acids 27 to 40) antiserum (DC35, 1:2,500), rabbit anti-erlin 2 (a kind gift from Dr. Stephen Robbins, 1:500), rabbit anti-calnexin (StressGen Biotechnologies, Victoria, BC, Canada; 1:500 or 1:5,000), rabbit anti-calreticulin (Affinity Bioreagents, Golden, CO; 1:1,000), goat anti-Cox 2 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:200), rabbit anti-fatty acid coenzyme A ligase 4 (FACL4, Abgent, 1:100), goat anti-Golgin 97 (Molecular Probes, Eugene, OR; 1:100), mouse anti-GRP75/mHSP70 (StressGen,1:2,500 or 1:5,000), rabbit anti-GRP78/BiP (StressGen, 1:500), mouse anti-Hexokinase 1 (Santa Cruz, 1:100), rabbit anti-HSP60 (GeneTex, Irvine, CA; 1:2,000), mouse anti-Membrin (StressGen, 1:150), rabbit anti-Mfn1 (Santa Cruz, 1:100), mouse anti-Mfn2 (AbCam, Cambridge, MA; 1:200), rabbit anti-PACS-2 (GeneTex, 1:500), goat anti-phosphoglycerate mutase (PGM) 5 (Santa Cruz, 1:100), rabbit anti-Prohibitin (GeneTex, 1:500), mouse anti-Sig-1R (Santa Cruz, 1:200 or 1:250), mouse antivoltage dependent anion channel (VDAC) (AbCam, 1:100 or 1:200), or mouse anti-␣-tubulin (Sigma, 1:2,000) and with the corresponding horseradish peroxidase-conjugated secondary antibody (1:2,500 or 1:5,000).…”

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

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Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Zhang

Williamson

Wong

et al. 2011

Molecular & Cellular Proteomics

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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 mitochondriaassociated 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

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Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

Section: Silac-labeling Of Hffs-primarymentioning

confidence: 99%

See 2 more Smart Citations

Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Zhang

Williamson

Wong

et al. 2011

Molecular & Cellular Proteomics

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“…As the SS is the first part of the polypeptide to emerge from the ribosome, it is likely that the nascent protein will be initially cotranslationally translocated into the ER lumen. From here, US9 could then be delivered to mitochondria through the mitochondrionassociated membranes, as is the case for vMIA (Bozidis et al, 2008;Colberg-Poley et al, 2000). Unlike vMIA, US9 does not contain an N-terminal bipartite signal and may anchor to the MOM through the C-terminal MLS.…”

Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis

Mandic

Miller

Coulter

et al. 2009

Journal of General Virology

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The human cytomegalovirus (CMV) US2-US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2-US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-a, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection. INTRODUCTIONHuman cytomegalovirus (CMV) is a very prevalent betaherpesvirus that poses serious medical concerns in immunocompromised individuals . The ability to evade or modulate host immune responses, coupled with the capacity to establish latency after primary infection, is at the basis of the tremendous success of CMV as a human pathogen.The US2-US11 genomic region has been shown to be dispensable for viral replication in primary human fibroblasts (HF) (Jones & Muzithras, 1992;Kollert-Jons et al., 1991), and to encode five endoplasmic reticulum (ER)-resident glycoproteins, US2, US3, US6, US10 and US11, that disrupt antigen presentation by the major histocompatibility complex (MHC) class I and class II proteins (Lin et al., 2007). Despite sharing some very limited homology with US6, US10 and US11, US9 does not bind to or reduce MHC cell surface levels (Hegde et al., 2002(Hegde et al., , 2006Pereira et al., 1995). US9 was originally described as a member of the US6 family of proteins, which includes the products of the US6-US11 genes. These proteins contain markedly hydrophobic sequences at both the N-and C-termini, and were predicted to insert into cellular membranes (Weston & Barrell, 1986). US9 intracellular localization was initially examined in nonpolarized Madin-Darby canine kidney cells constitutively expressing a C-terminal-tagged ...

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Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

et al. 2015

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Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV‐infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. © 2015 by John Wiley & Sons, Inc.

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Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells

Cited by 35 publications

References 54 publications

Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

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