Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system.
Johanna Maarit KoivistoNina Rødtness PoulsenBenedikte Stoklund LarsenM G M WeibullAmelie SteinFabio DoroJakob Rahr WintherKresten Lindorff-LarsenMartin WillemoësPublished in: The Biochemical journal (2022)
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.
Keyphrases
- escherichia coli
- antiretroviral therapy
- hiv positive
- hiv infected
- copy number
- human immunodeficiency virus
- hiv testing
- amino acid
- hepatitis c virus
- structural basis
- hiv aids
- klebsiella pneumoniae
- wild type
- men who have sex with men
- dna binding
- induced apoptosis
- genome wide
- transcription factor
- staphylococcus aureus
- sars cov
- dna methylation
- gene expression
- south africa
- working memory
- multidrug resistant
- signaling pathway