Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

Heaslet, H.; Lin, Y.-C.; Tam, K.; Torbett, Bruce E.; Elder, John H.; Stout, C.D.

doi:10.1186/1742-4690-4-1

Cited by 28 publications

(29 citation statements)

References 46 publications

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“…Using the NL4-3 strain as a template, protease mutants containing substitutions at these positions were constructed, then expressed and purified as described previously. 15 …”

Section: Major Drug Resistance Mutations Destabilize Hiv Proteasementioning

confidence: 99%

Accessory Mutations Maintain Stability in Drug-Resistant HIV-1 Protease

Chang

Torbett

2011

Journal of Molecular Biology

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The underlying mechanisms driving the evolution of drug resistance in HIV are only partially understood. We investigated the evolutionary cost of the major resistance mutations in HIV-1 protease in terms of protein stability. The accumulation of resistance mutations destabilizes the protease, limiting further adaptation. From an analysis of clinical isolates, we identified specific accessory mutations that were able to restore the stability of the protease or even increase it beyond the wild-type baseline. Resistance mutations were also found to decrease the activity of HIV protease near neutral pH values, while incorporating stabilizing mutations improved the enzyme’s pH tolerance. These findings help to explain the prevalence of mutations located far from the active site and underscore the importance of protein stability during the evolution of drug resistance in HIV.

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“…Using the NL4-3 strain as a template, protease mutants containing substitutions at these positions were constructed, then expressed and purified as described previously. 15 …”

Section: Major Drug Resistance Mutations Destabilize Hiv Proteasementioning

confidence: 99%

Accessory Mutations Maintain Stability in Drug-Resistant HIV-1 Protease

Chang

Torbett

2011

Journal of Molecular Biology

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“…The purified inclusion bodies were solubilized in 7.5 M urea containing 20 mM Tris, 5 mM EDTA pH 8, and insoluble material was removed by centrifugation at 8000 rev min À1 followed by filtration through a 0.45 mm membrane. The clarified solution was subsequently purified by ion-exchange chromatography as described previously (Heaslet et al, 2007). Briefly, the solution was mixed with Whatman DE52 anion exchanger, incubated for 30 min and filtered.…”

Section: Cloning Expression and Purificationmentioning

confidence: 99%

“…Data were collected on Stanford Synchrotron Radiation Lightsource beamline 11-1 (Soltis et al, 2008), integrated with MOSFLM (Leslie, 1999) and scaled with SCALA (Winn et al, 2011). The structures were solved by molecular replacement using MOLREP (Winn et al, 2011) with the 12s FIV PR structure (Heaslet et al, 2007) as a search model. Models were fitted to electron-density maps with MIFit (McRee, 1999) and were refined with REFMAC (Murshudov et al, 2011).…”

Section: Crystallization and Structure Determinationmentioning

confidence: 99%

“…The DRV-bound structure is also analyzed in terms of five mutants that do not support infectivity (Lin et al, 2010). Four additional mutants present in a non-infectious 12s FIV PR (Heaslet et al, 2007) are analyzed. The role of the mutants in affecting interactions with DRV and LPV is correlated with binding constants and the structures are compared with the corresponding wild-type HIV PR complexes (Tie et al, 2004;Stoll et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

Lin

Perryman

Olson

et al. 2011

Acta Crystallogr D Biol Cryst

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A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC(50) values in the nanomolar range.

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“…PR inclusion bodies were obtained, clarified, and solubilized in 7.5 M of urea containing 20 mM Tris-HCl, pH 8.0, and 5 mM EDTA. The PR was subsequently purified and refolded by ion-exchange chromatography and fast protein liquid chromatography as described before (10,28).…”

Section: Methodsmentioning

confidence: 99%

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

Lin

Happer

Elder

2013

J Virol

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An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/ N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC 50 s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99 82 A, I59 50 V, and V37 32 I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC 50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.

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Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

Abstract: We have obtained the 1.7

Cited by 28 publications

References 46 publications

Accessory Mutations Maintain Stability in Drug-Resistant HIV-1 Protease

Accessory Mutations Maintain Stability in Drug-Resistant HIV-1 Protease

Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

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