{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T20:22:08Z","timestamp":1781295728592,"version":"3.54.1"},"reference-count":17,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T00:00:00Z","timestamp":1734912000000},"content-version":"vor","delay-in-days":22,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain\u2010of\u2010function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS\u2010CoV\u20102, MERS\u2010CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. \u00a9 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol<\/jats:bold>: Using VSV as a platform for directed protease evolution<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Dose response assay with TCID<jats:sub>50<\/jats:sub> readout<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: A pipeline for high\u2010throughput VSV sequencing<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Rescue of VSV<\/jats:p>","DOI":"10.1002\/cpz1.70074","type":"journal-article","created":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T10:34:51Z","timestamp":1734950091000},"source":"Crossref","is-referenced-by-count":2,"title":["Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution"],"prefix":"10.1002","volume":"4","author":[{"given":"Francesco","family":"Costacurta","sequence":"first","affiliation":[{"name":"Institute of Virology Medical University of Innsbruck Innsbruck Austria"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7415-0211","authenticated-orcid":false,"given":"Stefanie","family":"Rauch","sequence":"additional","affiliation":[{"name":"Institute of Virology Medical University of Innsbruck Innsbruck Austria"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorothee","family":"von Laer","sequence":"additional","affiliation":[{"name":"Institute of Virology Medical University of Innsbruck Innsbruck Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8148-9490","authenticated-orcid":false,"given":"Emmanuel","family":"Heilmann","sequence":"additional","affiliation":[{"name":"Institute of Virology Medical University of Innsbruck Innsbruck Austria"},{"name":"Biological and Environmental Science and Engineering Division KAUST Thuwal Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,12,23]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.73.5.1504"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.73.2.442"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biologicals.2019.09.008"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giab008"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/v11110989"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010020\u201066942\u20106"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s44298\u2010024\u201000028\u20102"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty191"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1006\/smvy.1997.0105"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.7.5.651\u2010662.1971"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.70062"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978\u20100\u201012\u2010801238\u20103.95729\u20105"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0163962"},{"key":"e_1_2_9_15_1","first-page":"155","volume-title":"Nature Reviews Microbiology","author":"V'kovski P.","year":"2021"},{"key":"e_1_2_9_16_1","volume-title":"Microorganisms","author":"Wang M.","year":"2024"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gks918"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2006.02.006"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70074","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,12,27]],"date-time":"2024-12-27T07:54:43Z","timestamp":1735286083000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70074"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12]]},"references-count":17,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2024,12]]}},"alternative-id":["10.1002\/cpz1.70074"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70074","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,12]]},"article-number":"e70074"}}