BACKGROUND: Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. RESULTS: Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. CONCLUSION: In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• apoptóza genetika   MeSH
• buněčné linie MeSH
• CD4-pozitivní T-lymfocyty metabolismus   MeSH
• fragmentace DNA MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• HIV infekce genetika   metabolismus   MeSH
• HIV-1 genetika   metabolismus   MeSH
• HIV-proteasa genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein X asociovaný s bcl-2 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• AKIP1 protein, human MeSH Prohlížeč
• BAX protein, human MeSH Prohlížeč
• HIV-proteasa MeSH
• jaderné proteiny MeSH
• mitochondriální proteiny MeSH
• nádorový supresorový protein p53 MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• protein X asociovaný s bcl-2 MeSH
• TP53 protein, human MeSH Prohlížeč

UNLABELLED: We report enzymologic, thermodynamic and structural analyses of a series of six clinically derived mutant HIV proteases (PR) resistant to darunavir. As many as 20 mutations in the resistant PRs decreased the binding affinity of darunavir by up to 13 000-fold, mostly because of a less favorable enthalpy of binding that was only partially compensated by the entropic contribution. X-ray structure analysis suggested that the drop in enthalpy of darunavir binding to resistant PR species was mostly the result of a decrease in the number of hydrogen bonds and a loosening of the fit between the inhibitor and the mutated enzymes. The favorable entropic contribution to darunavir binding to mutated PR variants correlated with a larger burial of the nonpolar solvent-accessible surface area upon inhibitor binding. We show that even very dramatic changes in the PR sequence leading to the loss of hydrogen bonds with the inhibitor could be partially compensated by the entropy contribution as a result of the burial of the larger nonpolar surface area of the mutated HIV PRs. DATABASE: Atomic coordinates and structure factors for the crystal structures PRwt-DRV and PRDRV2-DRV complex have been deposited in the Protein Data Bank under accession codes 4LL3 and 3TTP, respectively. STRUCTURED DIGITAL ABSTRACT: • PR and PR bind by x-ray crystallography (View interaction).

• Klíčová slova
• HIV protease inhibitors, X‐ray crystallography, enthropic contribution, isothermal titration calorimetry, resistance mutation,
• MeSH
• darunavir MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu * MeSH
• sulfonamidy chemie   farmakologie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• darunavir MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• sulfonamidy MeSH

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.

• MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 chemie   účinky léků   enzymologie   genetika   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• inzerční mutageneze * MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• organofosfonáty analýza   MeSH
• sekvence aminokyselin MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• organofosfonáty MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč

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
• buněčné linie MeSH
• genové produkty gag - virus lidské imunodeficience genetika   MeSH
• genové produkty pol - virus lidské imunodeficience genetika   MeSH
• geny gag MeSH
• geny pol MeSH
• HEK293 buňky MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 genetika   izolace a purifikace   fyziologie   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy terapeutické užití   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• peptidové fragmenty genetika   MeSH
• virová léková rezistence genetika   MeSH
• virová nálož MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• gag protein p1, Human immunodeficiency virus MeSH Prohlížeč
• genové produkty gag - virus lidské imunodeficience MeSH
• genové produkty pol - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p2 gag peptide, Human immunodeficiency virus 1 MeSH Prohlížeč
• peptidové fragmenty 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
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 účinky léků   izolace a purifikace   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• darunavir MeSH
• genové produkty env - virus lidské imunodeficience MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• polyproteiny MeSH
• sulfonamidy 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
• alanin metabolismus   MeSH
• Escherichia coli genetika   MeSH
• HIV-proteasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   metabolismus   farmakologie   MeSH
• katalýza MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• Lopinavir MeSH
• molekulární modely MeSH
• náchylnost k nemoci * MeSH
• pyrimidinony chemie   metabolismus   farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   chemie   izolace a purifikace   MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin * MeSH
• virová léková rezistence genetika   MeSH
• vodíková vazba MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• Lopinavir MeSH
• pyrimidinony MeSH
• rekombinantní proteiny MeSH

HIV protease (PR) is a prime target for rational anti-HIV drug design. We have previously identified icosahedral metallacarboranes as a novel class of nonpeptidic protease inhibitors. Now we show that substituted metallacarboranes are potent and specific competitive inhibitors of drug-resistant HIV PRs prepared either by site-directed mutagenesis or cloned from HIV-positive patients. Molecular modeling explains the inhibition profile of metallacarboranes by their unconventional binding mode.

• MeSH
• HIV-1 účinky léků   enzymologie   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• kovy chemie   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• mutace genetika   MeSH
• sloučeniny boru chemie   farmakologie   MeSH
• virová léková rezistence účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• kovy MeSH
• sloučeniny boru MeSH

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

• MeSH
• dimerizace MeSH
• genetická variace * MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 účinky léků   enzymologie   genetika   MeSH
• HIV-proteasa účinky léků   genetika   imunologie   MeSH
• imunoglobuliny - fragmenty imunologie   farmakologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• monoklonální protilátky imunologie   farmakologie   MeSH
• mutace MeSH
• rekombinantní proteiny imunologie   farmakologie   MeSH
• virová léková rezistence genetika   MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• immunoglobulin Fv MeSH Prohlížeč
• imunoglobuliny - fragmenty MeSH
• inhibitory HIV-proteasy MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny 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
• buněčné linie MeSH
• chemické modely MeSH
• difrakce rentgenového záření MeSH
• HIV-1 enzymologie   genetika   fyziologie   MeSH
• HIV-proteasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• inhibitory reverzní transkriptasy chemie   MeSH
• inzerční mutageneze * MeSH
• katalýza MeSH
• kinetika MeSH
• konsenzuální sekvence MeSH
• látky proti HIV terapeutické užití   MeSH
• ledviny cytologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• replikace viru MeSH
• RNA virová analýza   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV MeSH
• rekombinantní proteiny MeSH
• RNA virová MeSH

Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that cleaves viral polyproteins into mature proteins, thus leading to the formation of infectious particles. Protease inhibitors (PIs) are successful virostatics. However, their efficiency is compromised by antiviral resistance. In the PR sequence of viral variants resistant to the PI nelfinavir, the mutations D30N and L90M appear frequently. However, these two mutations are seldom found together in vivo, suggesting that there are two alternative evolutionary pathways leading to nelfinavir resistance. Here we analyze the proteolytic activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1 PRs harboring the D30N and L90M mutations alone and in combination with other compensatory mutations. Vitality values obtained for recombinant mutant proteases and selected PR inhibitors confirm the crucial role of mutations in positions 30 and 90 for nelfinavir resistance. The combination of the D30N and L90M mutations significantly increases the enzyme vitality in the presence of nelfinavir, without a dramatic decrease in the catalytic efficiency of the recombinant enzyme. Crystal structures, molecular dynamics simulations, and calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M) were used to augment our kinetic data. Calorimetric analysis revealed that the entropic contribution to the mutant PR/nelfinavir interaction is less favorable than the entropic contribution to the binding of nelfinavir by wild-type PR. This finding is supported by the structural data and simulations; nelfinavir binds most strongly to the wild-type protease, which has the lowest number of protein-ligand hydrogen bonds and whose structure exhibits the greatest degree of fluctuation upon inhibitor binding.

• MeSH
• aktivace enzymů MeSH
• HIV-1 enzymologie   genetika   MeSH
• HIV-proteasa chemie   genetika   MeSH
• inhibitory HIV-proteasy chemie   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• nelfinavir chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• nelfinavir MeSH