{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:18:28Z","timestamp":1785460708698,"version":"3.56.0"},"reference-count":60,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2022,2,19]],"date-time":"2022-02-19T00:00:00Z","timestamp":1645228800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000945","name":"Pittsburgh Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000945","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010554","name":"Clinical and Translational Science Institute, University of Pittsburgh","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100010554","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001083","name":"DSF Charitable Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100001083","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Medical Virology"],"published-print":{"date-parts":[[2022,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The ongoing COVID\u201019 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome\u00a0coronavirus 2 (SARS\u2010CoV\u20102) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS\u2010CoV\u20102 minigenome replicon systems. First, we constructed the IVT\u2010CoV2\u2010Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT\u2010CoV2\u2010Rep in a variety of cell lines, which could be completely abolished by known SARS\u2010CoV\u20102 replication inhibitors. Our data also suggest\u00a0that the transient phenotype of IVT\u2010CoV2\u2010Rep is not due to host innate antiviral responses. In addition, we have developed a DNA\u2010launched replicon BAC\u2010CoV2\u2010Rep, which supports the in\u2010cell transcription of a replicon mRNA as initial replication template. The BAC\u2010CoV2\u2010Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT\u2010CoV2\u2010Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC\u2010CoV2\u2010Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS\u2010CoV\u20102 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.<\/jats:p>","DOI":"10.1002\/jmv.27650","type":"journal-article","created":{"date-parts":[[2022,2,9]],"date-time":"2022-02-09T10:06:20Z","timestamp":1644401180000},"page":"2438-2452","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Construction and characterization of two SARS\u2010CoV\u20102 minigenome replicon systems"],"prefix":"10.1002","volume":"94","author":[{"given":"Hu","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douglas K.","family":"Fischer","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahiro","family":"Shuda","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick S.","family":"Moore","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6194-1742","authenticated-orcid":false,"given":"Shou\u2010Jiang","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zandrea","family":"Ambrose","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7146-916X","authenticated-orcid":false,"given":"Haitao","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA"},{"name":"Cancer Virology Program, Hillman Cancer Center University of Pittsburgh Medical Center Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,2,19]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2012-7"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2008-3"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-0695-z"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-021-00573-0"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.27491"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-00459-7"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2020.02.001"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2020.12.010"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.26234"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-00468-6"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-virology-100114-055218"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.REV120.013930"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13578-021-00643-z"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.coviro.2014.04.007"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00491-8"},{"issue":"5","key":"e_1_2_9_17_1","first-page":"657","article-title":"The hepatitis C virus replicon system and its application to molecular studies","volume":"4","author":"Pietschmann T","year":"2001","journal-title":"Curr Opin Drug Discov Devel"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2020.03.010"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2006.10.016"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2025866118"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-82055-0"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02754-20"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.abj8430"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2020.104974"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2020.04.004"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00687-21"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.76.24.13001-13014.2002"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00869-16"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4222"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-020-00372-8"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.130502"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-484-5_13"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.22.12683-12688.2004"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-020-0282-0"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-22580-8"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-18233-x"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11095-020-02849-1"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008737"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1038\/s12276-021-00592-0"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2021107826"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2020.108234"},{"key":"e_1_2_9_42_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.79.5.2689-2699.2005"},{"key":"e_1_2_9_43_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.2.10.955-961.1968"},{"issue":"6","key":"e_1_2_9_44_1","first-page":"2279","article-title":"Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis","volume":"6","author":"Mosca JD","year":"1986","journal-title":"Mol Cell Biol"},{"key":"e_1_2_9_45_1","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.2008\/002097-0"},{"key":"e_1_2_9_46_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01317496"},{"key":"e_1_2_9_47_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.73.12.9891-9898.1999"},{"key":"e_1_2_9_48_1","doi-asserted-by":"publisher","DOI":"10.1038\/nri2314"},{"key":"e_1_2_9_49_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2022643118"},{"key":"e_1_2_9_50_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-291X(03)01318-4"},{"key":"e_1_2_9_51_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-61579-x"},{"key":"e_1_2_9_52_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2005.04.002"},{"key":"e_1_2_9_53_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.72.121801.161720"},{"key":"e_1_2_9_54_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-6481-9_2"},{"key":"e_1_2_9_55_1","doi-asserted-by":"publisher","DOI":"10.3390\/v12060598"},{"key":"e_1_2_9_56_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2014.05.026"},{"key":"e_1_2_9_57_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2294-9"},{"key":"e_1_2_9_58_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216-010-3821-6"},{"key":"e_1_2_9_59_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.10.004"},{"key":"e_1_2_9_60_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-020-0511-8"},{"key":"e_1_2_9_61_1","doi-asserted-by":"publisher","DOI":"10.1038\/313812a0"}],"container-title":["Journal of Medical Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jmv.27650","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/jmv.27650","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jmv.27650","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,25]],"date-time":"2023-08-25T14:46:19Z","timestamp":1692974779000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.27650"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,19]]},"references-count":60,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2022,6]]}},"alternative-id":["10.1002\/jmv.27650"],"URL":"https:\/\/doi.org\/10.1002\/jmv.27650","archive":["Portico"],"relation":{},"ISSN":["0146-6615","1096-9071"],"issn-type":[{"value":"0146-6615","type":"print"},{"value":"1096-9071","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,19]]},"assertion":[{"value":"2022-01-22","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-02-03","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-02-19","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}