RESUMO
The virion-associated dUTPase activities of caprine arthritis-encephalitis virus (CAEV) and visna virus were determined by using an assay which measure the actual ability of the dUTPase to prevent the dUTP misincorporations into cDNA during reverse transcription. We showed that the CAEV molecular clone from the Cork isolate was dUTPase defective as a result of a single amino acid substitution. Using this point mutant and deletion mutants of CAEV as well as a deletion mutant of visna virus, we demonstrated that dUTPase-deficient viruses replicate similarly to wild-type viruses in dividing cells but show delayed replication in nondividing primary macrophages.
Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Pirofosfatases/metabolismo , Replicação Viral/fisiologia , Vírus Visna-Maedi/fisiologia , Animais , Vírus da Artrite-Encefalite Caprina/enzimologia , Sequência de Bases , Células Cultivadas , DNA Viral , Humanos , Macrófagos/citologia , Dados de Sequência Molecular , Mutação , Pirofosfatases/genética , Homologia de Sequência de Aminoácidos , Nucleotídeos de Timina/metabolismo , Vírus Visna-Maedi/enzimologiaRESUMO
PURPOSE: To investigate the potential use of polymeric nanoparticles for the delivery of antisense oligonucleotides in HIV-1-infected cell cultures. METHODS: Phosphorothioate oligonucleotides were encapsulated into poly (D,L-lactic acid) nanoparticles. Two models of infected cells were used to test the ability of nanoparticles to deliver them. HeLa P4-2 CD4+ cells, stably transfected with the beta-galactosidase reporter gene, were first used to evaluate the activity of the oligonucleotides on a single-round infection cycle. The acutely infected lymphoid CEM cells were then used to evaluate the inhibition of the viral production of HIV-1 by the oligonucleotides. RESULTS: The addition to infected CEM cells of nanoparticles containing gag antisense oligonucleotides in the nanomolar range led to strong inhibition of the viral production in a concentration-dependent manner. Similar results were previously observed in HeLa P4-2 CD4+ cells. Nanoparticle-entrapped random-order gag oligonucleotides had similar effects on reverse transcription. However, the reverse transcriptase activity of infected cells treated with nanomolar concentrations of free antisense and random oligonucleotides was not affected. CONCLUSIONS: These results suggest that poly (D,L-lactic acid) nanoparticles may have great potential as an efficient delivery system for oligonucleotides in HIV natural target cells, i.e., lymphocytic cells.
Assuntos
HIV-1/efeitos dos fármacos , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Composição de Medicamentos , Excipientes , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Células HeLa , Humanos , Indicadores e Reagentes , Ácido Láctico , Linfócitos/efeitos dos fármacos , Linfócitos/virologia , Microesferas , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Poliésteres , Polímeros , Transfecção , Replicação Viral/efeitos dos fármacosRESUMO
The importance of the virally encoded dUTPase for CAEV replication, invasiveness, pathogenesis, and genetic stability was investigated in goats infected by viruses with single point (DU-G) and deletion (DU-1) mutations of the dUTPase gene (DU gene). The DU gene was found to be dispensable for CAEV replication in vivo as judged by times taken to seroconvert, frequencies of viral isolation, and tissue distribution of viral RNAs. DU- reversion at week 34 in one of three goats infected with the single point mutant DU-G, however, suggested that the viral dUTPase confers some advantages for replication in vivo. Moreover, we show that dUTPase is necessary for the timely development of bilateral arthritic lesions of the carpus. Finally, dUTPase was shown to efficiently prevent accumulation of G-to-A transitions in the viral genome.
Assuntos
Vírus da Artrite-Encefalite Caprina/enzimologia , Infecções por Lentivirus/microbiologia , Pirofosfatases/deficiência , Animais , Vírus da Artrite-Encefalite Caprina/patogenicidade , Sequência de Bases , DNA Viral/genética , Genes Virais , Doenças das Cabras/microbiologia , Doenças das Cabras/patologia , Cabras/microbiologia , Infecções por Lentivirus/patologia , Dados de Sequência Molecular , Monócitos/microbiologia , Mutação Puntual , Provírus/genética , Membrana Sinovial/ultraestrutura , Distribuição Tecidual , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
During the early phase of the retroviral life cycle, only a fraction of internalized virions end up integrating their genome into the chromosome, even though the resulting proviruses are almost systematically expressed. Here, we reveal that incoming retroviral preintegration complexes trigger the exportin-mediated cytoplasmic export of the SWI/SNF component INI1 and of the nuclear body constituent PML. We further show that the HIV genome associates with these proteins before nuclear migration. In the presence of arsenic, PML is sequestered in the nucleus, and the efficiency of HIV-mediated transduction is markedly increased. These results unveil a so far unsuspected cellular response that interferes with the early steps of HIV replication.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/virologia , HIV/crescimento & desenvolvimento , Carioferinas , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição/metabolismo , Replicação Viral/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona , Citoplasma/metabolismo , Citoplasma/virologia , HIV/genética , Células HeLa , Humanos , Proteína da Leucemia Promielocítica , Proteína SMARCB1 , Transfecção , Proteínas Supressoras de Tumor , Proteína Exportina 1RESUMO
The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.