{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T11:41:04Z","timestamp":1789731664207,"version":"4.0.1"},"reference-count":48,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2020,9,25]],"date-time":"2020-09-25T00:00:00Z","timestamp":1600992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["F32-AI122456"],"award-info":[{"award-number":["F32-AI122456"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32-AI049820"],"award-info":[{"award-number":["T32-AI049820"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01-AI081759"],"award-info":[{"award-number":["R01-AI081759"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000861","name":"Burroughs Wellcome Fund","doi-asserted-by":"publisher","award":["Investigators in the Pathogenesis of Infectious Disease Award"],"award-info":[{"award-number":["Investigators in the Pathogenesis of Infectious Disease Award"]}],"id":[{"id":"10.13039\/100000861","id-type":"DOI","asserted-by":"publisher"}]},{"name":"UPMC Children\u2019s Hospital of Pittsburgh","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and\/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.<\/jats:p>","DOI":"10.3390\/v12101074","type":"journal-article","created":{"date-parts":[[2020,9,25]],"date-time":"2020-09-25T08:57:32Z","timestamp":1601024252000},"page":"1074","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6379-0965","authenticated-orcid":false,"given":"Nicholas J.","family":"Lennemann","sequence":"first","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA"},{"name":"Center for Microbial Pathogenesis, UPMC Children\u2019s Hospital of Pittsburgh, Pittsburgh, PA 15224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8309-5222","authenticated-orcid":false,"given":"Azia S.","family":"Evans","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA"},{"name":"Center for Microbial Pathogenesis, UPMC Children\u2019s Hospital of Pittsburgh, Pittsburgh, PA 15224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolyn B.","family":"Coyne","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA"},{"name":"Center for Microbial Pathogenesis, UPMC Children\u2019s Hospital of Pittsburgh, Pittsburgh, PA 15224, USA"},{"name":"Richard K. Mellon Institute for Pediatric Research, UPMC Children\u2019s Hospital of Pittsburgh, Pittsburgh, PA 15224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,9,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1016\/j.virol.2010.12.014","article-title":"Enterovirus infections of the central nervous system","volume":"411","author":"Rhoades","year":"2011","journal-title":"Virology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1097\/01.inf.0000237798.07462.32","article-title":"Neonatal enterovirus infections reported to the national enterovirus surveillance system in the United States","volume":"25","author":"Khetsuriani","year":"2006","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_3","first-page":"1","article-title":"Enterovirus surveillance\u2014United States, 1970\u20132005","volume":"55","author":"Khetsuriani","year":"2006","journal-title":"MMWR Surveill. Summ."},{"key":"ref_4","first-page":"223","article-title":"Pediatric group B coxsackievirus infections","volume":"Volume 323","author":"Romero","year":"2008","journal-title":"Current Topics in Microbiology and Immunology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"936","DOI":"10.1056\/NEJM199909233411302","article-title":"Neurologic complications in children with enterovirus 71 infection","volume":"341","author":"Huang","year":"1999","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1086\/376998","article-title":"Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: Roles of cytokines and cellular immune activation in patients with pulmonary edema","volume":"188","author":"Wang","year":"2003","journal-title":"J. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"32","DOI":"10.5501\/wjv.v2.i2.32","article-title":"Architecture and biogenesis of plus-strand RNA virus replication factories","volume":"2","author":"Paul","year":"2013","journal-title":"World J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Limpens, R.W., van der Schaar, H.M., Kumar, D., Koster, A.J., Snijder, E.J., van Kuppeveld, F.J., and Barcena, M. (2011). The transformation of enterovirus replication structures: A three-dimensional study of single and double-membrane compartments. MBio, 2.","DOI":"10.1128\/mBio.00166-11"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Nagy, P.D., Strating, J.R., and van Kuppeveld, F.J. (2016). Building viral replication organelles: Close encounters of the membrane types. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005912"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8953","DOI":"10.1128\/JVI.74.19.8953-8965.2000","article-title":"Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: An autophagy-like origin for virus-induced vesicles","volume":"74","author":"Suhy","year":"2000","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/0042-6822(87)90063-8","article-title":"Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography","volume":"160","author":"Bienz","year":"1987","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1016\/0042-6822(65)90001-2","article-title":"Electron microscopic study of the formation of poliovirus","volume":"26","author":"Dales","year":"1965","journal-title":"Virology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6576","DOI":"10.1128\/jvi.70.10.6576-6588.1996","article-title":"Cellular origin and ultrastructure of membranes induced during poliovirus infection","volume":"70","author":"Schlegel","year":"1996","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"van der Schaar, H.M., Melia, C.E., van Bruggen, J.A., Strating, J.R., van Geenen, M.E., Koster, A.J., Barcena, M., and van Kuppeveld, F.J. (2016). Illuminating the sites of enterovirus replication in living cells by using a split-GFP-tagged viral protein. MSphere, 1.","DOI":"10.1128\/mSphere.00104-16"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Melia, C.E., Peddie, C.J., de Jong, A.W.M., Snijder, E.J., Collinson, L.M., Koster, A.J., van der Schaar, H.M., van Kuppeveld, F.J.M., and Barcena, M. (2019). Origins of enterovirus replication organelles established by whole-cell electron microscopy. MBio, 10.","DOI":"10.1128\/mBio.00951-19"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1016\/j.cell.2010.03.050","article-title":"Viral reorganization of the secretory pathway generates distinct organelles for RNA replication","volume":"141","author":"Hsu","year":"2010","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1128\/JVI.01820-06","article-title":"Hijacking components of the cellular secretory pathway for replication of poliovirus RNA","volume":"81","author":"Belov","year":"2007","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1002\/j.1460-2075.1995.tb07071.x","article-title":"Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A","volume":"14","author":"Doedens","year":"1995","journal-title":"EMBO J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3207","DOI":"10.1242\/jcs.03483","article-title":"Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex","volume":"120","author":"Beske","year":"2007","journal-title":"J. Cell Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.tim.2004.12.005","article-title":"Enterovirus protein 2B po(u)res out the calcium: A viral strategy to survive?","volume":"13","author":"Melchers","year":"2005","journal-title":"Trends Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"13081","DOI":"10.1073\/pnas.1401437111","article-title":"Nonlytic viral spread enhanced by autophagy components","volume":"111","author":"Bird","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1136\/jcp.50.5.422","article-title":"Tissue preparation for immunocytochemistry","volume":"50","author":"Williams","year":"1997","journal-title":"J. Clin. Pathol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1242\/bio.019570","article-title":"PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins","volume":"5","author":"Kedziora","year":"2016","journal-title":"Biol. Open"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/nmeth.1855","article-title":"Immunolabeling artifacts and the need for live-cell imaging","volume":"9","author":"Schnell","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"14610","DOI":"10.1073\/pnas.1212379109","article-title":"Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro","volume":"109","author":"Schoggins","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1016\/j.antiviral.2012.12.010","article-title":"Development and characterization of a stable eGFP enterovirus 71 for antiviral screening","volume":"97","author":"Shang","year":"2013","journal-title":"Antivir. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1038\/nbt.1604","article-title":"Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system","volume":"28","author":"Jones","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"McFadden, M.J., Mitchell-Dick, A., Vazquez, C., Roder, A.E., Labagnara, K.F., McMahon, J.J., Silver, D.L., and Horner, S.M. (2018). A fluorescent cell-based system for imaging Zika virus infection in real-time. Viruses, 10.","DOI":"10.3390\/v10020095"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.jviromet.2014.10.010","article-title":"A plasmid-based reporter system for live cell imaging of dengue virus infected cells","volume":"211","author":"Medin","year":"2015","journal-title":"J. Virol. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.chom.2011.01.001","article-title":"Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers","volume":"9","author":"Coyne","year":"2011","journal-title":"Cell Host Microbe"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Mukherjee, A., Morosky, S.A., Delorme-Axford, E., Dybdahl-Sissoko, N., Oberste, M.S., Wang, T., and Coyne, C.B. (2011). The coxsackievirus B 3C protease cleaves MAVS and TRIF to attenuate host type I interferon and apoptotic signaling. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1001311"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.chom.2015.07.007","article-title":"RIP3 Regulates Autophagy and Promotes Coxsackievirus B3 Infection of Intestinal Epithelial Cells","volume":"18","author":"Harris","year":"2015","journal-title":"Cell Host Microbe"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Shu, Q., Lennemann, N.J., Sarkar, S.N., Sadovsky, Y., and Coyne, C.B. (2015). ADAP2 Is an interferon stimulated gene that restricts RNA virus entry. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1005150"},{"key":"ref_34","first-page":"e02147","article-title":"BPIFB3 regulates autophagy and coxsackievirus B replication through a noncanonical pathway independent of the core initiation machinery","volume":"5","author":"Morosky","year":"2014","journal-title":"MBio"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"788","DOI":"10.1038\/s41598-017-00909-y","article-title":"High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry","volume":"7","author":"Wortzel","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1093\/emboj\/20.1.240","article-title":"Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition","volume":"20","author":"Gustin","year":"2001","journal-title":"EMBO J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/S0092-8674(00)80724-2","article-title":"Brefeldin A: The advantage of being uncompetitive","volume":"97","author":"Chardin","year":"1999","journal-title":"Cell"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"van der Schaar, H.M., Rust, M.J., Chen, C., van der Ende-Metselaar, H., Wilschut, J., Zhuang, X., and Smit, J.M. (2008). Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog., 4.","DOI":"10.1371\/journal.ppat.1000244"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3010","DOI":"10.1091\/mbc.e11-02-0094","article-title":"Calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus B from polarized intestinal epithelial cells","volume":"22","author":"Bozym","year":"2011","journal-title":"Mol. Biol. Cell"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Kaufusi, P.H., Kelley, J.F., Yanagihara, R., and Nerurkar, V.R. (2014). Induction of endoplasmic reticulum-derived replication-competent membrane structures by West Nile virus non-structural protein 4B. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0084040"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/j.stem.2016.07.019","article-title":"Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy","volume":"19","author":"Liang","year":"2016","journal-title":"Cell Stem Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"19117","DOI":"10.1073\/pnas.0609353103","article-title":"Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores","volume":"103","author":"Goodhouse","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Bozym, R.A., Morosky, S.A., Kim, K.S., Cherry, S., and Coyne, C.B. (2010). Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells. PLoS Pathog., 6.","DOI":"10.1371\/journal.ppat.1001135"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"903","DOI":"10.1242\/jcs.195263","article-title":"Mouse Stbd1 is N-myristoylated and affects ER-mitochondria association and mitochondrial morphology","volume":"130","author":"Demetriadou","year":"2017","journal-title":"J. Cell Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1556","DOI":"10.1091\/mbc.e09-12-1002","article-title":"Yip1A structures the mammalian endoplasmic reticulum","volume":"21","author":"Dykstra","year":"2010","journal-title":"Mol. Biol. Cell"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1083\/jcb.110.5.1501","article-title":"Membrane biogenesis during B cell differentiation: Most endoplasmic reticulum proteins are expressed coordinately","volume":"110","author":"Wiest","year":"1990","journal-title":"J. Cell Biol."},{"key":"ref_47","first-page":"4078","article-title":"ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery","volume":"127","author":"Schuck","year":"2014","journal-title":"J. Cell Sci."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Bernales, S., McDonald, K.L., and Walter, P. (2006). Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. 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