During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1' active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.

• MeSH
• Cell Line MeSH
• gag Gene Products, Human Immunodeficiency Virus genetics   MeSH
• pol Gene Products, Human Immunodeficiency Virus genetics   MeSH
• Genes, gag MeSH
• Genes, pol MeSH
• HEK293 Cells MeSH
• HIV Infections drug therapy   virology   MeSH
• HIV-1 genetics   isolation & purification   physiology   MeSH
• HIV Protease chemistry   genetics   metabolism   MeSH
• HIV Protease Inhibitors therapeutic use   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• Peptide Fragments genetics   MeSH
• Drug Resistance, Viral genetics   MeSH
• Viral Load MeSH
• Antiretroviral Therapy, Highly Active MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• gag protein p1, Human immunodeficiency virus MeSH Browser
• gag Gene Products, Human Immunodeficiency Virus MeSH
• pol Gene Products, Human Immunodeficiency Virus MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• p2 gag peptide, Human immunodeficiency virus 1 MeSH Browser
• Peptide Fragments MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• Darunavir MeSH
• env Gene Products, Human Immunodeficiency Virus metabolism   MeSH
• gag Gene Products, Human Immunodeficiency Virus metabolism   MeSH
• HIV Infections virology   MeSH
• HIV-1 drug effects   isolation & purification   MeSH
• HIV Protease chemistry   genetics   metabolism   MeSH
• HIV Protease Inhibitors pharmacology   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• DNA Mutational Analysis MeSH
• Polyproteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Amino Acid Substitution MeSH
• Sulfonamides pharmacology   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Drug Resistance, Viral * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Darunavir MeSH
• env Gene Products, Human Immunodeficiency Virus MeSH
• gag Gene Products, Human Immunodeficiency Virus MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Browser
• Polyproteins MeSH
• Sulfonamides MeSH

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

• MeSH
• Alanine metabolism   MeSH
• Escherichia coli genetics   MeSH
• HIV Protease chemistry   genetics   isolation & purification   metabolism   MeSH
• HIV Protease Inhibitors chemistry   metabolism   pharmacology   MeSH
• Catalysis MeSH
• Kinetics MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Lopinavir MeSH
• Models, Molecular MeSH
• Disease Susceptibility * MeSH
• Pyrimidinones chemistry   metabolism   pharmacology   MeSH
• Recombinant Proteins antagonists & inhibitors   chemistry   isolation & purification   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Substitution * MeSH
• Drug Resistance, Viral genetics   MeSH
• Hydrogen Bonding MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• Lopinavir MeSH
• Pyrimidinones MeSH
• Recombinant Proteins MeSH

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

• MeSH
• Cell Line MeSH
• Models, Chemical MeSH
• X-Ray Diffraction MeSH
• HIV-1 enzymology   genetics   physiology   MeSH
• HIV Protease chemistry   genetics   isolation & purification   metabolism   MeSH
• Reverse Transcriptase Inhibitors chemistry   MeSH
• Mutagenesis, Insertional * MeSH
• Catalysis MeSH
• Kinetics MeSH
• Consensus Sequence MeSH
• Anti-HIV Agents therapeutic use   MeSH
• Kidney cytology   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Recombinant Proteins chemistry   isolation & purification   metabolism   MeSH
• Virus Replication MeSH
• RNA, Viral analysis   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Drug Resistance, Viral * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• HIV Protease MeSH
• Reverse Transcriptase Inhibitors MeSH
• Anti-HIV Agents MeSH
• Recombinant Proteins MeSH
• RNA, Viral MeSH

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

• MeSH
• Aspartic Acid Endopeptidases chemistry   MeSH
• Boranes chemical synthesis   chemistry   pharmacology   MeSH
• HIV Protease chemistry   MeSH
• HIV Protease Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Crystallography, X-Ray MeSH
• Drug Design * MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Endopeptidases MeSH
• Boranes MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• p16 protease, Human immunodeficiency virus 2 MeSH Browser