UNLABELLED: HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. IMPORTANCE: Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

• MeSH
• HIV-1 fyziologie   MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny viru HIV metabolismus   MeSH
• proteolýza MeSH
• sestavení viru * MeSH
• uvolnění viru z buňky * MeSH
• virologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny viru HIV MeSH

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.

• Klíčová slova
• HIV-1 maturation, STED nanoscopy, native virus imaging, super-resolution microscopy,
• MeSH
• genové produkty gag - virus lidské imunodeficience chemie   MeSH
• HIV infekce MeSH
• HIV-1 * MeSH
• lidé MeSH
• peptidy MeSH
• proteolýza * MeSH
• tomografie elektronová * MeSH
• virion * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag - virus lidské imunodeficience MeSH
• Nano-1 peptide MeSH Prohlížeč
• peptidy 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

With the worldwide number of human immunodeficiency virus positive patients stagnant and the increasing emergence of viral strains resistant to current treatment, the development of novel anti-human immunodeficiency virus drug candidates is a perpetual quest of medicinal chemists. Herein, we report a novel group of diarylpyrimidines, non-nucleoside reverse transcriptase inhibitors, which represents an important class of current anti-human immunodeficiency virus therapy. Series of diarylpyrimidines containing o, o-difluorophenyl (A-arm), 4-cyanophenylamino (B-arm), and a small substituent (e.g. NH2, OMe) at positions 2, 4, and 6 of the pyrimidine ring were prepared. The A-arm was modified in the para position (F or OMe) and linked to the central pyrimidine core with a variable spacer (CO, O, NH). Antiviral activities of 20 compounds were measured against wild type human immunodeficiency virus-1 and mutant reverse transcriptase strains (K103N, Y181C) using a cytoprotection assay. To the most promising structural motives belong the o, o-difluoro- p-methoxy A-arm in position 4, and the amino group in position 6 of pyrimidine. Single digit nanomolar activities with no significant toxicity (CC50 > 17,000 nM) were found for compounds 35 (EC50 = 2 nM), 37 (EC50 = 3 nM), and 13 (EC50 = 4 nM) having O, NH, and CO linkers, respectively.

• Klíčová slova
• Diarylpyrimidine, etravirine, human immunodeficiency virus, non-nucleoside reverse transcriptase inhibitor, rilpivirine,
• MeSH
• buněčné linie MeSH
• HIV reverzní transkriptasa antagonisté a inhibitory   MeSH
• HIV-1 účinky léků   enzymologie   MeSH
• inhibitory reverzní transkriptasy chemická syntéza   chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• látky proti HIV chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie s uhlíkem 13C MeSH
• mikrobiální testy citlivosti MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• pyrimidiny chemická syntéza   chemie   farmakologie   MeSH
• vyvíjení léků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• HIV reverzní transkriptasa MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV MeSH
• pyrimidiny MeSH
• reverse transcriptase, Human immunodeficiency virus 1 MeSH Prohlížeč

CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

• MeSH
• buněčné linie MeSH
• cyklohexany MeSH
• fúze buněk MeSH
• genom virový MeSH
• genotyp MeSH
• genové produkty env - virus lidské imunodeficience genetika   MeSH
• HEK293 buňky MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 fyziologie   MeSH
• látky proti HIV farmakologie   MeSH
• lidé MeSH
• maravirok MeSH
• pilotní projekty MeSH
• receptory CCR5 genetika   MeSH
• receptory CXCR4 genetika   MeSH
• receptory HIV antagonisté a inhibitory   metabolismus   MeSH
• RNA virová genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• senzitivita a specificita MeSH
• triazoly MeSH
• tropismus virů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CXCR4 protein, human MeSH Prohlížeč
• cyklohexany MeSH
• genové produkty env - virus lidské imunodeficience MeSH
• látky proti HIV MeSH
• maravirok MeSH
• receptory CCR5 MeSH
• receptory CXCR4 MeSH
• receptory HIV MeSH
• RNA virová MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• triazoly MeSH
• URA3 protein, S cerevisiae MeSH Prohlížeč

OBJECTIVE: Late presentation of the patients with human immunodeficiency virus (HIV) infection is associated with less favourable treatment responses, more accelerated clinical progression, and a higher mortality risk. Although HIV prevalence is low in Turkey, it is steadily increasing and the information about late presentation among HIV-positives is limited. We aimed to analyze the status of late presentation among HIV-positive patients in Turkey. METHODS: All newly diagnosed HIV/AIDS patients from 2003 to 2016 were enrolled in this study by five dedicated centres in Istanbul, Turkey. Demographic data, CD4+ counts, and HIV RNA were collected from medical records and were transferred to a HIV database system. Late pre- sentation was defined as presentation for care with a CD4 count < 350 cells/mm3 or presentation with an AIDS-defining event, regardless of the CD4 cell count. A medical literature search was done for the analysis of late presentation in Turkey. RESULTS: The cohort included 1,673 patients (1,440 males, median age 35 years). Among them, 847 (50.6%) had an early diagnosis, with a CD count of more than 350 cells/mm3. The remaining 826 were late presenters. Among late presenters, 427 (25.5% of all, 51.7% of late presenters) presented with advanced HIV disease. Late presenters were more elderly and less educated. The gender seemed comparable between groups. Late presentation was more likely among married patients. Early presenters were more likely among homosexuals, those diagnosed in screening studies, and in lower HIV-RNA viral load category. There has been a decreasing trend among late presenters in 2011-2016 when compared to 2003-2011 period. CONCLUSION: Current data suggest that half of HIV-infected patients present late in Turkey. In our cohort, those presented late were more elderly, less educated, married and had heterosexual intercourse. On admission, late presenters had more HIV-related diseases and were more likely in higher HIV-RNA category. In the cohort, men having sex with men were less likely late presenters. Efforts to reduce the proportion of late presentation are essential for almost every country. The countries should identify the risk factors of late presentation and should improve early diagnosis and presentation for HIV care.

• Klíčová slova
• acquired immunodeficiency syndrome, human immunodeficiency virus infection, late presentation,
• MeSH
• dospělí MeSH
• heterosexualita statistika a číselné údaje   MeSH
• HIV infekce * diagnóza   epidemiologie   MeSH
• lidé MeSH
• opožděná diagnóza * MeSH
• počet CD4 lymfocytů metody   MeSH
• rizikové faktory MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Turecko MeSH

The Roche Cobas TaqScreen MPX Test v1 - multiplex reverse transcription-real time (MPX RT-Real Time) PCR, performed on Cobas s201 for HCV RNA, HBV DNA, HIV-1 RNA /group M and O/, and HIV-2 RNA was introduced as a supplement to the currently used imunoanalysis method for blood donor´s testing (Abbott CMIA - chemiluminescent microparticle imunoassay, performed on Architect i2000 for anti-HCV, hepatitis B surface antigen (HBsAg)), anti-HIV-1 /group M and O/, anti-HIV-2 and p24 HIV). The results of study could provide valuable arguments to support the discussion about the NAT implementation into the standards of blood donor´s testing in the Czech Republic. Two groups of samples were tested. In the first one, 5074 samples from consecutive blood donors, and in the second one, 5 repository preseroconverted samples from repeat blood donors, who were subsequently confirmed positive for Viral Hepatitis and/or HIV/AIDS by the National Reference Laboratory (NRL), were tested. One sample was found reactive by chemiluminescent microparticle immuno assay (CMIA) and nucleic acid test (NAT) (confirmed HBV-positive in NRL), 31 samples were CMIA-only reactive (15 anti-HCV, 4 HBsAg, 12 anti-HIV/p24, all confirmed negative in NRL) and one pool (6 samples) was found reactive (further individual NAT was negative for all samples) in the first group of samples. One sample was NAT-only reactive (confirmed HCV-positive in NRL) in the second group of samples. Our study confirmed that screening of infectious markers using NAT can reduce the risk of transmitting the monitored infections by blood transfusion in the Czech Republic, even as a country with currently good epidemiological situation.

• MeSH
• Hepacivirus genetika   izolace a purifikace   MeSH
• hepatitida B diagnóza   epidemiologie   virologie   MeSH
• hepatitida C diagnóza   epidemiologie   virologie   MeSH
• HIV infekce diagnóza   epidemiologie   virologie   MeSH
• HIV-1 genetika   izolace a purifikace   MeSH
• HIV-2 genetika   izolace a purifikace   MeSH
• lidé MeSH
• virus hepatitidy B genetika   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.

• MeSH
• COS buňky MeSH
• HIV-1 účinky léků   MeSH
• inhibitory HIV-proteasy chemická syntéza   chemie   farmakologie   MeSH
• molekulární modely MeSH
• stereoizomerie MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory HIV-proteasy MeSH

The inhibitory effect of azidothymidine (AZT) on HIV replication in the promonocytic cell line U937 was investigated. After infection with HIV-1/LAV virus, U937 cells were cultured for prolonged period in the presence of the drug at a final concentrations of 20 mumol/l or 50 mumol/l, respectively. The antiviral activity was determined according to the inhibition of viral reverse transcriptase activity, and of viral antigen production (immunofluorescence assay). We conclude that virus production was not efficiently influenced during long term passage even at high drug concentrations.

• MeSH
• chemická deprese MeSH
• difúzní velkobuněčný B-lymfom patologie   MeSH
• HIV antigeny biosyntéza   MeSH
• HIV reverzní transkriptasa MeSH
• HIV-1 účinky léků   fyziologie   MeSH
• inhibitory reverzní transkriptasy MeSH
• lidé MeSH
• monocyty účinky léků   mikrobiologie   MeSH
• nádorové buňky kultivované účinky léků   mikrobiologie   MeSH
• replikace viru účinky léků   MeSH
• zidovudin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• HIV antigeny MeSH
• HIV reverzní transkriptasa MeSH
• inhibitory reverzní transkriptasy MeSH
• zidovudin MeSH

A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

• MeSH
• CD4-pozitivní T-lymfocyty virologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• genetická transkripce * MeSH
• HIV imunologie   fyziologie   MeSH
• kohortové studie MeSH
• kokultivační techniky MeSH
• kultivované buňky MeSH
• lidé MeSH
• RNA-polymerasa II metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• RNA-polymerasa II MeSH