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Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.<\/jats:p>","DOI":"10.1042\/bcj20210767","type":"journal-article","created":{"date-parts":[[2022,1,28]],"date-time":"2022-01-28T14:56:47Z","timestamp":1643381807000},"page":"479-501","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an <i>Escherichia coli<\/i> genetic selection system"],"prefix":"10.1042","volume":"479","author":[{"given":"Johanna Maarit","family":"Koivisto","sequence":"first","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nina R\u00f8dtness","family":"Poulsen","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benedikte Stoklund","family":"Larsen","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M.G.M.","family":"Weibull","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amelie","family":"Stein","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabio","family":"Doro","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jakob Rahr","family":"Winther","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4750-6039","authenticated-orcid":false,"given":"Kresten","family":"Lindorff-Larsen","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin","family":"Willemo\u00ebs","sequence":"additional","affiliation":[{"name":"From the Linderstr\u00f8m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maal\u00f8es Vej 5, Copenhagen 2200N, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2022,2,17]]},"reference":[{"key":"2022022512554186600_BCJ-2021-0767C1","doi-asserted-by":"publisher","first-page":"577","DOI":"10.1089\/aid.1989.5.577","article-title":"Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease","volume":"5","year":"1989","journal-title":"AIDS Res. 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