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

Potts, Karen E.; Smidt, Mary L.; Tucker, Simon P.; Stiebel, T.R.; McDonald, Joseph J.; Stallings, William C.; Bryant, Martin L.

doi:10.1177/095632029700800508

Cited by 3 publications

(7 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Proviral DNA of the protease gene was amplified by PCR and was used for sequencing or cloning. DNAs from strains 89-959 and SF162 were cloned and sequenced as described by Potts et al (21). Briefly, infected cells were lysed and extracted with phenol-chloroform-isoamyl alcohol, and the DNA was precipitated.…”

Section: Methodsmentioning

confidence: 99%

“…Construction of proviral clones and characterization of mutated HIV-1. Oligonucleotide-mediated site-directed mutagenesis of the protease gene and the production of infectious mutated viruses was done by the methods described by Potts et al (21). Mutated protease genes were made in HXB2gpt2 (HXB2) by using the Altered Sites In Vitro Mutagenesis System (Promega).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Smidt

Potts

Tucker

et al. 1997

Antimicrob Agents Chemother

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Smidt

Potts

Tucker

et al. 1997

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…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). Progeny viruses were derived from the mutated proviral DNA and were used to establish virus stocks (termed "recombinant variants").…”

mentioning

confidence: 82%

“…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.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Tucker¹,

Stiebel²,

Potts³

et al. 1998

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The frequency of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants in virus populations not previously exposed to drug was determined in vitro by using HIV-1RF and the protease inhibitor SC-55389A. Two variants with single mutations responsible for drug resistance (V82A and N88S) were quantifiably isolated after only one round of replication, yielding a crude frequency estimate of at least 1 SC-55389A-resistant variant per 3.5 × 105wild-type infectious units.

show abstract

Virus population dynamics, fitness variations and the control of viral disease: an update

Domingo

Más

Yuste

et al. 2001

Progress in Drug Research 57

View full text Add to dashboard Cite

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

Abstract: In vitro sequential selection and characterization of human immunodeficiency virus type 1 variants with reduced sensitivity to hydroxyethylurea protease inhibitors 447 contributions of the observed substitutions to drug resistance is presented in molecular modelling studies.

Cited by 3 publications

References 37 publications

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

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

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

Virus population dynamics, fitness variations and the control of viral disease: an update

Contact Info

Product

Resources

About