In vitro Sequential Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Reduced Sensitivity to Hydroxyethylurea Protease Inhibitors

In vitro resistance to the human immunodeficiency virus (HIV) protease inhibitors SC-52151 and SC-55389A was evaluated in an in vitro sequential selection scheme. HIV_RF variants were selected for reduced sensitivity to SC-52151 and subsequently passaged in both SC-52151 and a structurally different hydroxyethylurea protease inhibitor, SC-55389A, to select for dual-resistant virus. SC-52151 selection alone resulted in a 23-fold reduction in virus sensitivity whereas selection in both inhibitors resulted in 34- and eightfold reductions in virus sensitivity to SC-52151 and SC-55389A, respectively. Sequence analysis of the protease gene revealed that SC-52151 -resistant virus had a Gly to Val substitution at residue 48 (G48V) and, in 58% of subclones, an accompanying Val to Ala substitution at residue 82 (V82A). Dual-resistant virus had both G48V and V82A substitutions present and, in the majority of subclones, an lle to Thr and/or Leu to Pro substitution at residues 54 and 63, respectively. Drug susceptibility assays with limiting dilution-cloned HIV_RFR (G48V/V82A) and HIV_RFRR (G48V/154T/L63P/V82A) viruses demonstrated moderate to high-level cross-resistance to additional structurally non-related protease inhibitors. Recombinant HIV_HXB2 proviral clones with G48V, L63P and V82A substitutions showed that one active site mutation was permissible, but the presence of both G48V and V82A substitutions together significantly reduced infectious virus production. Insight into the contributions of the observed substitutions to drug resistance is presented in molecular modelling studies.