A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. 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.
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February 2022
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Representative fluorescence micrograph of fibrin amyloid microclots from an individual with Long COVID [232]
Research Article|
February 17 2022
Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system
Johanna Maarit Koivisto;
Johanna Maarit Koivisto
*
Supervision, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Nina Rødtness Poulsen;
Nina Rødtness Poulsen
*
Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Benedikte Stoklund Larsen;
Benedikte Stoklund Larsen
Formal analysis, Investigation, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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M.G.M. Weibull;
M.G.M. Weibull
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Amelie Stein;
Amelie Stein
Formal analysis, Visualization, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Fabio Doro;
Fabio Doro
Visualization, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Jakob Rahr Winther;
Jakob Rahr Winther
Supervision, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
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Kresten Lindorff-Larsen
;
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
Correspondence: Kresten Lindorff-Larsen (lindorff@bio.ku.dk) or Martin Willemoës (willemoes@bio.ku.dk)
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Martin Willemoës
Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing
From the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200N, Denmark
Correspondence: Kresten Lindorff-Larsen (lindorff@bio.ku.dk) or Martin Willemoës (willemoes@bio.ku.dk)
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Publisher: Portland Press Ltd
Received:
November 02 2021
Revision Received:
January 07 2022
Accepted:
January 28 2022
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2022
Biochem J (2022) 479 (4): 479–501.
Article history
Received:
November 02 2021
Revision Received:
January 07 2022
Accepted:
January 28 2022
Citation
Johanna Maarit Koivisto, Nina Rødtness Poulsen, Benedikte Stoklund Larsen, M.G.M. Weibull, Amelie Stein, Fabio Doro, Jakob Rahr Winther, Kresten Lindorff-Larsen, Martin Willemoës; Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system. Biochem J 25 February 2022; 479 (4): 479–501. doi: https://doi.org/10.1042/BCJ20210767
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