RESUMO
The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis, HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment BssHII255-EcoRI4183) genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level.
Assuntos
Colo/virologia , Genes gag/genética , Genes pol/genética , HIV-1/fisiologia , Replicação Viral/genética , Linhagem Celular , Colo/citologia , DNA Viral/biossíntese , Produtos do Gene gag/genética , Genoma Viral , Transcriptase Reversa do HIV , HIV-1/genética , HIV-1/patogenicidade , Humanos , Cinética , Regiões Promotoras Genéticas/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Ribonuclease H/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Lectins with specificity for terminal mannose residues and anti-mannan antibodies neutralize HIV-1 infection in vitro. This is assumed to be caused by binding of the agents to the viral glycoproteins. In this study we show that one such agent, the Galanthus nivalis lectin (GNA), also blocks infection at the target cell level. To explore the effect of GNA on HIV infection we used the two HIV-1 isolates LAV and NDK, representing in the first case a prototype virus and in the latter case a highly cytopathic virus, which spreads preferentially via cell-to-cell contact. MT-4 cells were used as target cells and infection was determined from the occurrence of syncytia. Cell-to-cell infection was studied with CEM cells persistently infected with the two virus isolates. GNA, at concentrations in the nanogram per milliliter range, neutralized the HIV-1 isolates LAV, NDK, and MN as well as HIV-2ROD. Pretreatment of cells with the lectin, before addition of virus, or of infected cells, also blocked infection. This effect was more pronounced with HIV-1NDK than with HIV-1LAV. Mannosidase treatment of the target cells abolished the GNA effect on HIV-1NDK infection. It is concluded that GNA inhibits infection of several HIV isolates. It neutralizes infection by binding to the virion but also blocks infection at the target cell level. The latter effect may be different for different virus isolates. Mannosyl residuals at the cell surface are targets for GNA modulation of infection with the cytopathic HIV-1NDK. These do not represent essential virus receptors.
Assuntos
Antivirais/farmacologia , HIV/efeitos dos fármacos , Lectinas/farmacologia , Lectinas de Ligação a Manose , Linhagem Celular , Galanthus , HIV/patogenicidade , Infecções por HIV/prevenção & controle , Humanos , Lectinas de Plantas , Virulência/efeitos dos fármacosRESUMO
The HIV1-PAR strain, isolated from the cerebrospinal fluid of an HIV1-seropositive man suffering from encephalopathy, replicated well in cord blood lymphocytes, poorly in peripheral blood mononuclear cells, and to different levels in blood-derived macrophage (BDM) cultures prepared from different blood donors. In marked contrast to its replication in primocultures, it did not grow in CEM and U937 cell lines. HIV1-PAR production in BDM was inhibited by more than 90% after treatment with OKT4A or 13B8.2 monoclonal antibodies (mAb) binding to adjacent epitopes of the D1 domain of the CD4 molecules. A lower but significant inhibitory effect was observed after BDM treatment with BL4 and OKT4 mAb, directed to the D2 and D3 domain of the CD4 molecule, respectively. The entire HIV1-PAR envelope glycoprotein gene was amplified by polymerase chain reaction and sequenced. The deduced amino acid sequence of HIV1-PAR gp160 revealed the presence of 847 amino acids and 86% homology with the HIV1 LAV virus prototype. An alignment of the amino acid sequence of the envelope glycoprotein of HIV1-PAR and HIV1-LAV showed that the differences were mostly clustered within the five variable regions. Five CD4-binding domains, the gp120/gp41 cleavage site, the putative gp41 fusion domain and 21 out of the 22 cysteine residues were conserved in both isolates. The results further confirm the macrophage-tropic character of the HIV1-PAR virus.
Assuntos
Antivirais/fisiologia , Antígenos CD4/fisiologia , Produtos do Gene env/química , HIV-1/fisiologia , Leucócitos Mononucleares/microbiologia , Precursores de Proteínas/química , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos CD4/imunologia , Linhagem Celular , Proteína gp120 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV , Soropositividade para HIV/líquido cefalorraquidiano , HIV-1/química , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Replicação ViralRESUMO
The human colonic adenocarcinoma cell line HT29 can be infected with various isolates of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). In some cases, the virus was able to perform its complete cycle of replication as demonstrated by the persistent production of mature viral particles in the cell-free culture supernatant. We have cultured HT29 cells chronically infected with the replicative strain HIV1-NDK in a chemically defined serum-free medium. Under these conditions, the cells were able to maintain a high level of viral replication, as demonstrated by reverse transcriptase activities and in situ hybridization studies. By indirect immunofluorescence labeling and electron microscopy, we observed that serum starvation was associated with the differentiation of HIV-1-infected HT29 cells into mucous-secreting cells resembling epithelial goblet cells of the colonic mucosa. These mucous-secreting cells, which accounted for 50% of the overall population, produced mature particles of HIV through their apical membrane in the vicinity of mucous granules. These data suggest that HIV-infected goblet cells in the colonic mucosa may produce the virus in the colorectal lumen; this could explain the route of transmission of HIV in the case of anal intercourse.
Assuntos
HIV-1/fisiologia , Replicação Viral , Adenocarcinoma , Neoplasias do Colo , Meios de Cultura Livres de Soro , Epitélio/metabolismo , Imunofluorescência , Transcriptase Reversa do HIV , HIV-1/ultraestrutura , Humanos , Hibridização In Situ , Microscopia Eletrônica , DNA Polimerase Dirigida por RNA/análise , Células Tumorais CultivadasRESUMO
Pretreatment of human colon epithelial cells HT29 by recombinant gamma interferon (IFN)-gamma was found to protect the cells from infection with various isolates of human immunodeficiency virus (HIV)-1 and HIV-2, as assessed by co-cultivation with human T lymphoblastoid cells and gene amplification by polymerase chain reaction technique. Additionally, IFN-gamma induced a dose-dependent inhibition of HIV-1 and HIV-2 production in chronically infected HT29 cells. In situ hybridization studies demonstrated that IFN-treated cells were still able to synthesize viral messenger ribonucleic acid. However, the expression of the p24 product of the gag gene was markedly decreased after IFN treatment as demonstrated by radio-immunoprecipitation assay. Taken together, these data suggested that the cytokine acted at the post-translational level by inhibiting the processing of structural viral proteins. It is concluded from this study that IFN-gamma has a potent anti-HIV effect on epithelial gastrointestinal cells.
Assuntos
Colo/microbiologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Interferon gama/farmacologia , Células Cultivadas , Epitélio/microbiologia , HIV-1/fisiologia , HIV-2/fisiologia , Humanos , Proteínas Recombinantes , Proteínas Estruturais Virais/biossínteseRESUMO
The human colon epithelial cell line HT29 can be infected by selected strains of the human immunodeficiency virus (HIV) [9]. In the present study, it is shown that tumor necrosis factor-alpha (TNF-alpha) is a potent stimulator of HIV replication in chronically infected differentiated HT29 cells, but not in undifferentiated cells. The polarity of HIV production upon TNF-alpha stimulation has been studied in polarized monolayers of differentiated HT29 cells grown on porous-bottomed dishes. It is shown that the cytokine induced a dramatic increase of HIV production through the two opposite sides of the monolayer, i.e. the apical and basolateral plasma membrane domains. The effect of TNF-alpha was mainly localized at the level of viral mRNA synthesis as demonstrated by in situ hybridization. These data support the concept that cytokines released as a result of intestinal inflammatory responses could promote HIV replication and contribute to the gastrointestinal disease in HIV-infected patients.
Assuntos
HIV/fisiologia , Intestinos/microbiologia , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacos , Síndrome da Imunodeficiência Adquirida/etiologia , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Hibridização In Situ , Intestinos/citologia , RNA Viral/biossíntese , DNA Polimerase Dirigida por RNA/biossíntese , Fatores de Tempo , Transcrição GênicaRESUMO
The macrophage-tropic virus HIV1-PAR, isolated from cerebrospinal fluid of HIV1-seropositive man, induced cytopathic effect accompanied by different magnitude of the virus production in blood-derived macrophages (BDM) obtained from different donors. HIV1-PAR-specific RNA was detected by in situ hybridization in 15 and 66% of BDM producing low and high levels of virus, respectively. In contrast with HIV1-PAR, infection of BDM with two laboratory strains adapted to T-cell lines, HIV1-LAV prototype and HIV1-NDK, a Zairian virus that is highly cytopathic for T-lymphocytes, resulted in a low production of HIV1 p24gag in culture fluid. Expression of HIV1-LAV and HIV1-NDK RNA was detected by in situ hybridization in a maximum of 1% of macrophages. Only HIV1-NDK, and not HIV1-LAV, induced ultrastructural alterations in BDM. In contrast with a striking difference in the production of macrophage-tropic and T-lymphotropic viruses, no significant differences were found in the proportion of macrophages containing retrotranscribed genomes of HIV1-. HIV1 DNA was detected by in situ hybridization in 93, 100, and 80% of macrophages infected with HIV1-PAR, HIV1-LAV, and HIV1-NDK, respectively. A higher level of HIV1 DNA was detected by polymerase chain reaction in the BDM infected with HIV1-PAR than in that infected with HIV1-LAV and HIV1-NDK. The results indicate that both macrophage-tropic as well as T-lymphotropic viruses can enter and retrotranscribe their genomes in a vast majority of macrophages.
Assuntos
HIV-1/fisiologia , Macrófagos/microbiologia , DNA Viral/análise , Sangue Fetal , Proteína do Núcleo p24 do HIV/análise , Transcriptase Reversa do HIV , HIV-1/enzimologia , HIV-1/genética , HIV-1/ultraestrutura , Humanos , Leucócitos Mononucleares/microbiologia , Linfócitos/microbiologia , Macrófagos/ultraestrutura , Microscopia Eletrônica , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Provírus/genética , DNA Polimerase Dirigida por RNA/metabolismo , Replicação ViralRESUMO
We have studied the expression of the desmin gene, a muscle-specific intermediate filament protein in the granuloma cells of mouse liver infected with Schistosoma mansoni. In situ hybridization using a desmin DNA probe showed that fibroblastic cells in the granuloma strongly expressed desmin mRNAs, while in normal liver these cells did not express this mRNA to a detectable degree. The quantitative analysis of total RNAs demonstrated that the proportion of specific desmin mRNA increased from 14 to 18 weeks after infection and decreased at 20 weeks. The analysis of collagen gene expression indicated that the amount of type III collagen mRNAs was still increasing after 18 weeks from infection; in contrast, the amount of type I collagen mRNAs remained unchanged at that stage. A good correlation was observed between the detection of the specific mRNAs and the detection of both desmin and collagen molecules. Therefore, these data point to a coordinate induction of desmin and collagen gene expression during Schistosomal granuloma formation. They also suggest that the expression of the myofibroblast phenotype involves the induction of both genes.
Assuntos
Desmina/biossíntese , Regulação da Expressão Gênica , Granuloma/metabolismo , Hepatopatias Parasitárias/metabolismo , Esquistossomose mansoni/metabolismo , Animais , Northern Blotting , Feminino , Fibroblastos/metabolismo , Imunofluorescência , Granuloma/patologia , Hepatopatias Parasitárias/patologia , Camundongos , Hibridização de Ácido Nucleico , Esquistossomose mansoni/patologiaRESUMO
We have used a probe encoding for the human desmin gene to study the expression of the desmin gene in skeletal and smooth muscle by in situ hybridization. In human skeletal muscle, the results showed a strong and homogeneous level of desmin mRNA contrasting with the faintly immunostaining of the desmin protein. In smooth muscle cells of colon and uterus, in situ hybridization and immunofluorescence staining suggests that there are some cells which do not contain desmin. The optimal condition of desmin mRNA detection was in cryostat sections fixed with paraformaldehyde and in paraffin embedded tissue with the same fixative. The human desmin probe can be used as a marker of cell differentiation and a way to study the regulation of the expression of the desmin gene in pathological events.