{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T11:13:05Z","timestamp":1784718785223,"version":"3.55.0"},"reference-count":48,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2016,5,20]],"date-time":"2016-05-20T00:00:00Z","timestamp":1463702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31272534"],"award-info":[{"award-number":["31272534"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Key Technology R&amp;D Program in the Twelfth Five-Year Plan","award":["2013BAD20B02, 2013BAD19B09-2"],"award-info":[{"award-number":["2013BAD20B02, 2013BAD19B09-2"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Porcine circovirus type 2 (PCV2) induces autophagy via the 5\u2032 adenosine monophosphate-activated protein kinase (AMPK)\/extracellular signal-regulated kinase (ERK)\/tuberous sclerosis complex 2 (TSC2)\/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium\/calmodulin-dependent protein kinase kinase-beta (CaMKK\u03b2) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-\u03b3). CaMKK\u03b2 then activated both AMPK and calcium\/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.<\/jats:p>","DOI":"10.3390\/v8050135","type":"journal-article","created":{"date-parts":[[2016,5,21]],"date-time":"2016-05-21T19:24:37Z","timestamp":1463858677000},"page":"135","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Porcine Circovirus Type 2 Activates CaMMK\u03b2 to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium"],"prefix":"10.3390","volume":"8","author":[{"given":"Yuanxing","family":"Gu","sequence":"first","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baozhu","family":"Qi","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingshan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaowu","family":"Jiang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xian","family":"Zhang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoliang","family":"Li","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weihuan","family":"Fang","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2016,5,20]]},"reference":[{"key":"ref_1","first-page":"44","article-title":"Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome","volume":"39","author":"Ellis","year":"1998","journal-title":"Can. Vet. J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1146\/annurev-animal-031412-103720","article-title":"Porcine circovirus type 2 (PCV2): Pathogenesis and interaction with the immune system","volume":"1","author":"Meng","year":"2013","journal-title":"Ann. Rev. Anim. Biosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1006\/viro.2002.1733","article-title":"Transcriptional analysis of porcine circovirus type 2","volume":"305","author":"Cheung","year":"2003","journal-title":"Virology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.virusres.2011.10.012","article-title":"Porcine circovirus: Transcription and DNA replication","volume":"164","author":"Cheung","year":"2012","journal-title":"Virus Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"8135","DOI":"10.1128\/JVI.78.15.8135-8145.2004","article-title":"Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2","volume":"78","author":"Lekcharoensuk","year":"2004","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"7856","DOI":"10.1128\/JVI.00737-11","article-title":"The 2.3-angstrom structure of porcine circovirus 2","volume":"85","author":"Khayat","year":"2011","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1016\/j.virusres.2011.11.012","article-title":"Porcine circovirus type 2 explores the autophagic machinery for replication in pk-15 cells","volume":"163","author":"Zhu","year":"2012","journal-title":"Virus Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.virol.2014.07.051","article-title":"Porcine circovirus-2 capsid protein induces cell death in PK15 cells","volume":"468\u2013470","author":"Walia","year":"2014","journal-title":"Virology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"8262","DOI":"10.1128\/JVI.79.13.8262-8274.2005","article-title":"Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis","volume":"79","author":"Liu","year":"2005","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1420","DOI":"10.1128\/JVI.01443-12","article-title":"Identification and functional analysis of the novel orf4 protein encoded by porcine circovirus type 2","volume":"87","author":"He","year":"2013","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Lv, Q., Guo, K., Xu, H., Wang, T., and Zhang, Y. (2015). Identification of putative ORF5 protein of porcine circovirus type 2 and functional analysis of GFP-fused ORF5 protein. PloS ONE, 10.","DOI":"10.1371\/journal.pone.0134203"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Klausmann, S., Sydler, T., Summerfield, A., Lewis, F.I., Weilenmann, R., Sidler, X., and Brugnera, E. (2015). T-cell reprogramming through targeted CD4-coreceptor and t-cell receptor expression on maturing thymocytes by latent circoviridae family member porcine circovirus type 2 cell infections in the thymus. Emerg. Microbes Infect., 4.","DOI":"10.1038\/emi.2015.15"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1016\/j.vaccine.2016.01.005","article-title":"Latent porcine circovirus type 2-infected domestic pigs: A potential infection model for the effective development of vaccines against latent or chronic virus induced diseases","volume":"34","author":"Sydler","year":"2016","journal-title":"Vaccine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/ncb2979","article-title":"Cargo recognition and trafficking in selective autophagy","volume":"16","author":"Stolz","year":"2014","journal-title":"Nat. Cell Biol."},{"key":"ref_15","first-page":"193","article-title":"Membrane dynamics in autophagosome biogenesis","volume":"128","author":"Carlsson","year":"2015","journal-title":"J. Cell Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/j.chom.2009.05.016","article-title":"Autophagy, immunity, and microbial adaptations","volume":"5","author":"Deretic","year":"2009","journal-title":"Cell Host Microbe"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/j.virol.2015.03.042","article-title":"Viruses and the autophagy pathway","volume":"479\u2013480","author":"Jackson","year":"2015","journal-title":"Virology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"83","DOI":"10.3390\/cells2010083","article-title":"Divergent roles of autophagy in virus infection","volume":"2","author":"Chiramel","year":"2013","journal-title":"Cells"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/35036035","article-title":"The versatility and universality of calcium signalling","volume":"1","author":"Berridge","year":"2000","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/nrm1155","article-title":"Calcium signalling: Dynamics, homeostasis and remodelling","volume":"4","author":"Berridge","year":"2003","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"97","DOI":"10.2174\/13892037112139990033","article-title":"Ca2+-sensor proteins in the autophagic and endocytic traffic","volume":"14","author":"Ghislat","year":"2013","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1016\/j.ceca.2011.04.001","article-title":"A dual role for Ca(2+) in autophagy regulation","volume":"50","author":"Decuypere","year":"2011","journal-title":"Cell Calcium"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9100","DOI":"10.1074\/jbc.M109.060061","article-title":"AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism","volume":"285","author":"Vingtdeux","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.molcel.2006.12.009","article-title":"Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2","volume":"25","author":"Bastholm","year":"2007","journal-title":"Mol. Cell"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1016\/j.cellsig.2010.01.015","article-title":"AMPK-independent induction of autophagy by cytosolic Ca2+ increase","volume":"22","author":"Grotemeier","year":"2010","journal-title":"Cell. Signal."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1124\/mol.111.071761","article-title":"Ca2+\/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy","volume":"80","author":"Pfisterer","year":"2011","journal-title":"Mol. Pharmacol."},{"key":"ref_27","first-page":"207","article-title":"WIPI proteins: Essential PtdIns3P effectors at the nascent autophagosome","volume":"128","author":"Takacs","year":"2015","journal-title":"J. Cell Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1546","DOI":"10.1016\/j.febslet.2015.05.008","article-title":"Function of human WIPI proteins in autophagosomal rejuvenation of endomembranes?","volume":"589","author":"Muller","year":"2015","journal-title":"FEBS Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"497","DOI":"10.4161\/auto.27419","article-title":"Detection of WIPI1 mRNA as an indicator of autophagosome formation","volume":"10","author":"Tsuyuki","year":"2014","journal-title":"Autophagy"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3396","DOI":"10.1016\/j.febslet.2007.06.040","article-title":"Human WIPI-1 puncta-formation: A novel assay to assess mammalian autophagy","volume":"581","author":"Ruckerbauer","year":"2007","journal-title":"FEBS Lett."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ceca.2009.05.005","article-title":"Viral calciomics: Interplays between Ca2+ and virus","volume":"46","author":"Zhou","year":"2009","journal-title":"Cell Calcium"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2834","DOI":"10.3390\/v7062749","article-title":"The emerging roles of viroporins in ER stress response and autophagy induction during virus infection","volume":"7","author":"Fung","year":"2015","journal-title":"Viruses"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"18465","DOI":"10.1073\/pnas.1204652109","article-title":"Hepatitis B virus x protein targets the Bcl-2 protein CED-9 to induce intracellular ca2+ increase and cell death in Caenorhabditis elegans","volume":"109","author":"Geng","year":"2012","journal-title":"Proc. Nat. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"8721","DOI":"10.1128\/JVI.00144-10","article-title":"Virus-induced Ca2+ influx extends survival of West Nile virus-infected cells","volume":"84","author":"Scherbik","year":"2010","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"E3405","DOI":"10.1073\/pnas.1216539109","article-title":"Autophagy hijacked through viroporin-activated calcium\/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus replication","volume":"109","author":"Crawford","year":"2012","journal-title":"Proc. Nat. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1016\/j.rvsc.2012.04.003","article-title":"PCV2 induces apoptosis and modulates calcium homeostasis in piglet lymphocytes in vitro","volume":"93","author":"Lv","year":"2012","journal-title":"Res. Vet. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"12003","DOI":"10.1128\/JVI.01434-12","article-title":"Porcine circovirus type 2 induces autophagy via the AMPK\/ERK\/TSC2\/mTOR signaling pathway in PK-15 cells","volume":"86","author":"Zhu","year":"2012","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1038\/nmeth.1243","article-title":"A genetically encoded calcium indicator for chronic in vivo two-photon imaging","volume":"5","author":"Mank","year":"2008","journal-title":"Nat. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5","DOI":"10.4161\/auto.22379","article-title":"Recombinant protein rVP1 upregulates BECN1-independent autophagy, MAPK1\/3 phosphorylation and MMP9 activity via WIPI1\/WIPI2 to promote macrophage migration","volume":"9","author":"Liao","year":"2013","journal-title":"Autophagy"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1146\/annurev-physiol-030212-183750","article-title":"Mammalian phospholipase c","volume":"75","author":"Kadamur","year":"2013","journal-title":"Ann. Rev. Hysiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.4161\/cc.9.7.11109","article-title":"Viruses and the autophagy machinery","volume":"9","author":"Dreux","year":"2010","journal-title":"Cell Cycle"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1002\/rmv.687","article-title":"Viruses and the fuel sensor: The emerging link between AMPK and virus replication","volume":"21","author":"Mankouri","year":"2011","journal-title":"Rev. Med. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1016\/j.cmet.2015.05.005","article-title":"Beyond energy homeostasis: The expanding role of AMP-activated protein kinase in regulating metabolism","volume":"21","author":"Carling","year":"2015","journal-title":"Cell Metab."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1016","DOI":"10.1038\/ncb2329","article-title":"The AMPK signalling pathway coordinates cell growth, autophagy and metabolism","volume":"13","author":"Mihaylova","year":"2011","journal-title":"Nat. Cell Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ceca.2011.02.007","article-title":"Analysis of CaM-kinase signaling in cells","volume":"50","author":"Wayman","year":"2011","journal-title":"Cell Calcium"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"31658","DOI":"10.1074\/jbc.R112.356485","article-title":"Calcium\/calmodulin-dependent protein kinase kinase 2: Roles in signaling and pathophysiology","volume":"287","author":"Racioppi","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Moser, T.S., Schieffer, D., and Cherry, S. (2012). AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002661"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1002\/path.4201","article-title":"Inhibition of the LKB1-AMPK pathway by the Epstein-Barr virus-encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells","volume":"230","author":"Lo","year":"2013","journal-title":"J. Pathol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/8\/5\/135\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T19:24:16Z","timestamp":1760210656000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/8\/5\/135"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,5,20]]},"references-count":48,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2016,5]]}},"alternative-id":["v8050135"],"URL":"https:\/\/doi.org\/10.3390\/v8050135","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,5,20]]}}}