Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system
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Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system. / Koivisto, Johanna Maarit; Poulsen, Nina Rødtness; Larsen, Benedikte Stoklund; Weibull, M. G. M.; Stein, Amelie; Doro, Fabio; Winther, Jakob Rahr; Lindorff-Larsen, Kresten; Willemoës, Martin.
In: The Biochemical journal, Vol. 479, No. 4, 2022, p. 479-501.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system
AU - Koivisto, Johanna Maarit
AU - Poulsen, Nina Rødtness
AU - Larsen, Benedikte Stoklund
AU - Weibull, M. G. M.
AU - Stein, Amelie
AU - Doro, Fabio
AU - Winther, Jakob Rahr
AU - Lindorff-Larsen, Kresten
AU - Willemoës, Martin
N1 - Copyright 2022 The Author(s).
PY - 2022
Y1 - 2022
N2 - 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 substate 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.
AB - 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 substate 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.
U2 - 10.1042/BCJ20210767
DO - 10.1042/BCJ20210767
M3 - Journal article
C2 - 35089310
VL - 479
SP - 479
EP - 501
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 4
ER -
ID: 291220477