A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A

Abstract

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.

Understanding HIV resistance, fitness, replication capacity and compensation: targeting viral fitness as a therapeutic strategy

“…Asn88Ser also causes reduced sensitivity to BMS-232632 and SC-55389A [188,192]. The reduced fitness of viruses possessing the Asn88Ser mutation has been described [193][194][195]. Continued selection resulted in the appearance of compensatory mutations that resulted in increased fitness levels followed by the appearance of a mutation, Met46Leu, which further reduced fitness without effects on processing or drug sensitivity.…”

Estimate of the Frequency of Human Immunodeficiency Virus Type 1 Protease Inhibitor Resistance within Unselected Virus Populations In Vitro

“…The culture was divided into 960 subcultures in 10 96-well tissue culture plates (primary plates), which were incubated in the presence or absence of 0.5 g of SC-55389A/ml (2, 7). A 0.5-g/ml concentration of SC-55389A was used because standard dose-response assays (22) revealed that this concentration completely inhibited viral cytopathic effect (CPE) and supernatant RT activity above background. (All assays, including viral-titer assays, involved the identical cell line and virus stocks that were used in the drug selection experiment.…”

“…(All assays, including viral-titer assays, involved the identical cell line and virus stocks that were used in the drug selection experiment. TCID 50 assays were scored by CPE and RT on day 7 postinfection [22] and several days thereafter to ensure accurate titers.) The cultures were split 1:2 and were fed with medium containing fresh drug weekly.…”

“…Genomic DNA was also extracted from a sample of infected cells at this time. A region of pol encompassing the protease gene was amplified from the genomic DNA of virus-positive samples by PCR and sequenced by direct-cycle sequencing of the PCR product (18,22). To obtain quasiclonal viral stocks for dose-response assays, cell-free supernatants from the cocultures were subjected to three rounds of growth at limiting dilution in the absence of drug.…”

“…Sequencing of PCR-amplified proviral DNA derived from the drug-treated cultures confirmed the presence of nucleotide changes encoding amino acid substitutions in the protease which were absent from the wild-type controls grown in the absence of drug (Table 1). Of the changes observed, N88S (2 of 9) and V82A (2 of 9) have previously been correlated with resistance to SC-55389A (17,18,21,22). To confirm that the mutations observed were responsible for drug resistance, each was re-created by oligonucleotide site-directed mutagenesis in an HIV-1 HXB2 proviral clone (18,22).…”