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To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.<\/jats:p>","DOI":"10.1073\/pnas.1101515108","type":"journal-article","created":{"date-parts":[[2011,6,6]],"date-time":"2011-06-06T21:58:27Z","timestamp":1307397507000},"page":"10290-10295","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":39,"title":["Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals"],"prefix":"10.1073","volume":"108","author":[{"given":"Margaret","family":"Robinson","sequence":"first","affiliation":[{"name":"Gilead Sciences, Foster City, CA 94404"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Tian","sequence":"additional","affiliation":[{"name":"Gilead Sciences, Foster City, CA 94404"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"IV","given":"William E.","family":"Delaney","sequence":"additional","affiliation":[{"name":"Gilead Sciences, Foster City, CA 94404"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew E.","family":"Greenstein","sequence":"additional","affiliation":[{"name":"Gilead Sciences, Foster City, CA 94404"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2011,6,6]]},"reference":[{"key":"e_1_3_3_1_2","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/28.6.491"},{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.3000544"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-8278(07)61648-1"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2009.11.055"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01217-10"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.285.5424.110"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1114016"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1517\/13543784.17.3.303"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01149-07"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.49.5.2059-2069.2005"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M305041200"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2007.11.010"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkp328"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.14.7400-7409.2004"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00289-10"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1001118"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01004-06"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.49.10.4305-4314.2005"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01236-09"},{"key":"e_1_3_3_20_2","first-page":"25","article-title":"Mutations in the hepatitis C virus polymerase that increase RNA binding can confer resistance to cyclosporine A. 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