Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system

Abstract

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.

Analysis of mutational history of multidrug‐resistant genotypes with a mutagenetic tree model

“…Margerison et al 23 identified K55R associated with M46I/L, which is in line with our evolutionary model that presents M46I as an ancestor and M46L as a descendant of K55R. I66V and E34Q have previously been identified as resistance mutations during PI treatment 24–27 . Mutation V118I is known to be associated with resistance to the NRTI class 28 .…”

“…I66V and E34Q have previously been identified as resistance mutations during PI treatment. [24][25][26][27] Mutation V118I is known to be associated with resistance to the NRTI class. 28 It also plays a part in dual resistance to AZT and 3TC together with mutation E44D.…”