RESUMO
The present study was undertaken to examine structural features of L-chicoric acid (3) which are important for potency against purified HIV-1 integrase and for reported cytoprotective effects in cell-based systems. Through a progressive series of analogues, it was shown that enantiomeric D-chicoric acid (4) retains inhibitory potency against purified integrase equal to its L-counterpart and further that removal of either one or both carboxylic functionalities results in essentially no loss of inhibitory potency. Additionally, while two caffeoyl moieties are required, attachment of caffeoyl groups to the central linking structure can be achieved via amide or mixed amide/ester linkages. More remarkable is the finding that blockage of the catechol functionality through conversion to tetraacetate esters results in almost no loss of potency, contingent on the presence of at least one carboxyl group on the central linker. Taken as a whole, the work has resulted in the identification of new integrase inhibitors which may be regarded as bis-caffeoyl derivatives of glycidic acid and amino acids such as serine and beta-aminoalanine. The present study also examined the reported ability of chicoric acid to exert cytoprotective effects in HIV-infected cells. It was demonstrated in target and cell-based assays that the chicoric acids do not significantly inhibit other targets associated with HIV-1 replication, including reverse transcription, protease function, NCp7 zinc finger function, or replication of virus from latently infected cells. In CEM cells, for both the parent chicoric acid and selected analogues, antiviral activity was observable under specific assay conditions and with high dependence on the multiplicity of viral infection. However, against HIV-1- and HIV-2-infected MT-4 cells, the chicoric acids and their tetraacetylated esters exhibited antiviral activity (50% effective concentration (EC50) ranging from 1.7 to 20 microM and 50% inhibitory concentration (IC50) ranging from 40 to 60 microM).
Assuntos
Fármacos Anti-HIV/farmacologia , Ácidos Cafeicos , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , Succinatos/síntese química , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Estereoisomerismo , Relação Estrutura-Atividade , Succinatos/química , Succinatos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products. An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.
Assuntos
DNA Viral/sangue , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Humanos , Parvovirus B19 Humano/genética , Plasmídeos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND OBJECTIVES: A collaborative study was undertaken to establish a replacement for the current (1st) World Health Organization (WHO) hepatitis C virus (HCV) International Standard, 96/790. MATERIALS AND METHODS: Both the 1(st) International Standard and the replacement standard were prepared from the same starting material by diluting a high titre genotype 1a HCV isolate in pooled, human plasma. The only difference was that each standard was lyophilized in two, separate lyophilisation runs but under the same conditions. RESULTS: In the study to establish the 1st International Standard, no significant difference in potency was found between the material eventually designated as the 1st International Standard and that now selected as the 2nd International Standard. The present study also showed no significant differences between the materials stored at -20 degrees C and no evidence of degradation over 5 years. CONCLUSIONS: Material 96/798 was established as the 2nd HCV International Standard and assigned the same unitage as the 1st International Standard, i.e. 10(5) IU/ml (50,000 IU/vial).
Assuntos
Hepacivirus/genética , Hepatite C/diagnóstico , RNA Viral/normas , Viremia/diagnóstico , Virologia/normas , Primers do DNA/química , Testes Hematológicos , Hepacivirus/isolamento & purificação , Hepatite C/sangue , Humanos , Cooperação Internacional , Reação em Cadeia da Polimerase , RNA Viral/sangue , RNA Viral/isolamento & purificação , Padrões de Referência , Projetos de Pesquisa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Viremia/sangue , Organização Mundial da SaúdeRESUMO
Human immunodeficiency virus type 1 mutants that are resistant to inhibition by cyclosporins arise spontaneously in vitro during propagation in a HeLa-CD4+ cell line in the presence of a nonimmunosuppressive analog of cyclosporin A. Interestingly, the phenotype of all of the mutants examined is drug resistant and drug dependent, with both cyclosporin A and its analog. Four independently isolated mutants have been analyzed genetically by construction of recombinant proviruses in the NL4-3 parental strain background and subsequent testing of the chimeric viruses in HeLa cells. The cyclosporin-resistant, cyclosporin-dependent phenotype consistently transfers with a 1.3-kb fragment of gag, within which the four mutants share one of two possible single amino acid exchanges in a proline-rich stretch in the capsid domain of Pr55gag. These mutants provide the first evidence that mutations in human immunodeficiency virus type 1 gag confer resistance to cyclosporins; however, replication is conditional on the presence of the drug. In the T-cell line CEM, replication of the recombinant mutant viruses is also cyclosporin dependent. The drug-dependent replication in HeLa cells is stringent, and in the absence of cyclosporin only revertant viruses with the parental phenotype grow out of cultures infected with cyclosporin-dependent virus. In at least one isolate examined, the revertant phenotype appears to be due to suppressor mutations near the proline-rich region.
Assuntos
Ciclosporina/farmacologia , Genes gag , HIV-1/efeitos dos fármacos , Mutação , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Resistência a Medicamentos , HIV-1/genética , HIV-1/fisiologia , Células HeLa , Humanos , Dados de Sequência MolecularRESUMO
In addition to its role in receptor binding, the envelope glycoprotein of certain human immunodeficiency virus type 2 (HIV-2) isolates, including ROD10, exhibits a biological activity that enhances the release of HIV-2, HIV-1, and simian immunodeficiency virus particles from infected cells. The present study aims at better defining the functional domains involved in this biological activity. To this end, we have characterized the envelope protein of the ROD14 isolate of HIV-2, which, despite 95% homology with the ROD10 envelope at the amino acid level, is unable to enhance viral particle release. Site-directed mutagenesis showed that the presence of a truncation in the cytoplasmic tail of the ROD14 envelope was not responsible for the lack of activity, as previously reported for the HIV-2 ST isolate (G. D. Ritter, Jr., G. Yamshchikov, S. J. Cohen, and M. J. Mulligan, J. Virol. 70:2669-2673, 1996). Similarly, several modifications of the length of the ROD10 envelope cytoplasmic tail did not impair its ability to enhance particle release, suggesting that, in the case of the HIV-2 ROD isolate, particle release activity is not regulated by the length of the cytoplasmic tail.
Assuntos
Produtos do Gene env/fisiologia , Herpesvirus Humano 2/fisiologia , Vírion/fisiologia , Sequência de Aminoácidos , Citoplasma , Produtos do Gene env/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-AtividadeRESUMO
In this study, we present the first detection of a focus of tick-borne encephalitis (TBE) virus-infected ticks in Liechtenstein. The focus is located on a much-used forest path near Vaduz, the capital of the principality. The virus isolated is a representative of the Western subtype of the TBE virus. It is thus closely related to or identical with the other strains isolated in western Europe.
Assuntos
Encefalite Transmitida por Carrapatos/epidemiologia , Animais , Vetores Aracnídeos/microbiologia , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/transmissão , Feminino , Humanos , Liechtenstein/epidemiologia , Masculino , Camundongos , Carrapatos/microbiologiaRESUMO
There have been a few severe cases of tick-borne encephalitis in Liechtenstein during the last 20 years. To form a better idea of the risk of infection and the potential benefit of vaccination, a total of 311 sera from different cohorts were investigated for antibodies to tick-borne encephalitis. The mean seroprevalence found was 3.6% and was not higher even in persons who were active in professional forestry. The antibodies measured derived in all groups mainly from previous vaccination and in only 2 cases (0.6%) from natural infection. It is concluded that the risk of TbE infection in Liechtenstein is very low. Therefore, a reduction in cases would probably be achieved only by mass vaccination.
Assuntos
Encefalite Transmitida por Carrapatos/epidemiologia , Estudos Soroepidemiológicos , Anticorpos Antivirais/isolamento & purificação , Estudos de Coortes , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/imunologia , Humanos , Liechtenstein/epidemiologiaRESUMO
The cellular peptidyl-prolyl isomerase cyclophilin A is incorporated into human immunodeficiency virus type 1 virions via contacts with the proline-rich domain of the Gag polyprotein. Cyclosporine A and nonimmunosuppressive analogs bind with high affinity to cyclophilin A, compete with Gag for binding to cyclophilin A, and prevent incorporation of cyclophilin A into virions; in parallel with the disruption of cyclophilin A incorporation into virions, there is a linear reduction in the initiation of reverse transcription after infection of a T cell. Passage of human immunodeficiency virus type 1 in the presence of the drug selects one of two mutations, either of which alters the proline-rich domain of Gag and is sufficient to confer drug resistance on the cloned wild-type provirus. Neither mutation alters Gag's cyclophilin A-binding properties in vitro, and cyclophilin A incorporation into drug-resistant virions is effectively disrupted by cyclosporine A, indicating that the drug-resistant mutants do not require virion-associated cyclophilin A to initiate infection. That Gag's functional dependence on cyclophilin A can be differentiated genetically from its ability to bind cyclophilin A is further demonstrated by the rescue of a mutation precluding cyclophilin A packaging by a mutation conferring cyclosporine A resistance. These experiments demonstrate that, in addition to its ability to package cyclophilin A into virions, gag encodes the functional target of cyclophilin A.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Isomerases de Aminoácido/farmacologia , Proteínas de Transporte/farmacologia , Ciclosporina/farmacologia , Produtos do Gene gag/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Isomerases de Aminoácido/uso terapêutico , Sequência de Bases , Proteínas de Transporte/uso terapêutico , Células Cultivadas , Resistência a Medicamentos , Produtos do Gene gag/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Peptidilprolil IsomeraseRESUMO
The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.