RESUMEN

The HIV-1 wild-type Rev and the negative oligomerization-defective mutant RevM4 were fused to enhanced green fluorescent protein (EGFP) and the tetrameric red fluorescent protein (DsRed1) followed by examination of their intracellular localization and Rev activity. As previously shown, fusion of EGFP to Rev and RevM4 did not affect the biological activity. Fusion of DsRed1 to Rev inhibited activity, and interestingly, fusion of DsRed1 to RevM4 restored activity. Based on these results, a model is proposed suggesting how RevM4-DsRed1 is able to rescue Rev activity through oligomerization on the viral RNA.

Asunto(s)

VIH-1/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Fusión Artificial Génica , Células COS , Chlorocebus aethiops , Citoplasma/química , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Biológicos , Unión Proteica , ARN Viral/metabolismo , Proteína Fluorescente Roja

RESUMEN

To study functional aspects of the exon 1 encoded region of the human immunodeficiency virus type 1 Rev protein, the viral Tev protein which exhibits low Rev activity but lacks the rev exon 1 encoded region was examined. Neither Rev-Tev heteromer complex formation nor inhibition of Rev by an export deficient Tev mutant was observed. Insertion of the rev exon 1 encoded region into the Tev mutant allowed it to oligomerize with Rev and act as a trans-dominant negative mutant. This showed that the exon 1 encoded region of Rev is essential for oligomerization and that oligomerization is a prerequisite for trans-dominant inhibition.

Asunto(s)

Exones/genética , Productos del Gen rev/fisiología , VIH-1/genética , Supresión Genética/fisiología , Animales , Células CHO , Células COS , Cricetinae , Técnica del Anticuerpo Fluorescente , Productos del Gen rev/antagonistas & inhibidores , Genes Dominantes , Sustancias Macromoleculares , Mutación , Unión Proteica/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Four monoclonal antibodies (MAbs) recognizing HIV-1 reverse transcriptase (RT) were shown here to cross-react with the beta' subunit of Escherichia coli RNA polymerase (RNAP). The anti-RT MAbs bind to a peptide comprising residues 294-305 of the RT amino acid sequence. Computer analyses revealed sequence similarity between this peptide and two regions of the RNAP beta' subunit. MAb-binding studies using RT mutants suggested that the epitope is located to amino acids 652-663 of the beta' sequence. One of the MAbs which inhibited the polymerase activity of RT also mediated a dose dependent inhibition of the RNAP activity.

Asunto(s)

Reacciones Cruzadas/inmunología , ARN Polimerasas Dirigidas por ADN/inmunología , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Transcriptasa Inversa del VIH/inmunología , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Western Blotting , Catálisis/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/química , Relación Dosis-Respuesta a Droga , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Cinética , Datos de Secuencia Molecular , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Conformación Proteica , Subunidades de Proteína , Relación Estructura-Actividad

RESUMEN

The HIV-1 protein Rev regulates the cytoplasmic levels of incompletely spliced HIV-1 mRNAs. The plasmid pSVc21, which contains a HIV-1 provirus, was introduced into COS cells by transient transfection. Simultaneous detection of HIV-1 RNAs and Rev proteins produced in transfected cells was then performed in order to determine the relative distribution of these two components. HIV-1 RNAs and the Rev protein localized to the same areas of the nucleoplasm, implying that these locations represent sites where Rev interacts with its target RNAs. Using a monoclonal antibody targeted to the splicing factor SC-35 it was demonstrated that the sites where HIV-1 mRNAs and Rev were detected often contained weak anti-SC-35 staining, whereas little RNA and Rev were found in strongly labeled SC-35-containing speckles. The same distribution of HIV-1 RNAs relative to SC-35 was also seen in transfected HeLa cells and in primary human lymphocytes infected with HIV-1 primary isolates. In addition, transiently expressed intron-containing beta-globin RNAs were shown to distribute to weak anti-SC-35 staining in a manner similar to that of HIV-1 RNAs. The findings suggest that Rev and HIV-1 RNAs interact at putative sites of mRNA transcription and splicing.

Asunto(s)

Productos del Gen rev/metabolismo , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , ARN Viral/metabolismo , Ribonucleoproteínas , Animales , Anticuerpos Monoclonales , Sitios de Unión , Células COS , Núcleo Celular/metabolismo , Núcleo Celular/virología , Productos del Gen rev/inmunología , Globinas/genética , VIH-1/genética , Humanos , Hibridación Fluorescente in Situ , Intrones , Proteínas Nucleares/inmunología , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Factores de Empalme Serina-Arginina , Fracciones Subcelulares , Transfección , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

HeLa cells and HeLa cells expressing the HIV-1 regulatory protein Rev were immunostained for Rev and pre-mRNA processing factors and examined histographically by confocal laser scanning microscopy. Following short pulse-labelling with bromouridine tri-phosphate nascent RNA gave a granular nucleoplasmic staining increasing somewhat towards the periphery as did also the heterogeneous ribonucleoproteins (hnRNPs) A1 and particularly C1/C2, a distribution pattern which has not been described. The sm-antigen of the small ribonucleoprotein particle (snRNP) proteins U1, U2, U4/U6 and U5 stained the nucleoplasm diffusely in addition to speckles which co-localised with speckles of the non-snRNP splicing factor SC-35. Brominated RNA and the hnRNPs A1 and C1/C2 were to varying degrees excluded from the speckles. Rev concentrated in the nucleolus and often as a perinucleolar ring/zone. Rev also stained the nucleoplasm and cytoplasm without co-localising with the above-mentioned proteins or brominated RNA and was not enriched or excluded in SC-35 speckles. The nucleolar proteins B23 and C23, like Rev, gave primarily a perinucleolar ring and stained the nucleoplasm but did not otherwise co-localise with Rev or with nuclear proteins. Histographic recording of immunofluorescence images proved to be a valuable tool in the study of localisation of HIV-1 Rev and cellular components and of possible co-localisations. A parallel comparison of the subcellular patterns of pre-mRNA processing factors versus major nucleolar antigens is new and suggests that the factors are not strictly separated in the nucleoplasm.

Asunto(s)

Productos del Gen rev/análisis , VIH-1/química , Proteínas Nucleares/análisis , Animales , Bromodesoxiuridina/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunohistoquímica , Ratones , ARN Mensajero/metabolismo , ARN Viral/análisis , Transfección , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) Rev is a small RNA-binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N- and C-terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988-2996) predicting that a helix-loop-helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Productos del Gen rev/química , VIH-1/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Bromelaínas/metabolismo , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Mapeo Epitopo , Productos del Gen rev/inmunología , Productos del Gen rev/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

Asunto(s)

Núcleo Celular/metabolismo , Productos del Gen rev/química , Productos del Gen rev/metabolismo , VIH-1/metabolismo , Animales , Western Blotting , Células COS , Citoplasma/metabolismo , Productos del Gen rev/biosíntesis , VIH-1/genética , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.

Asunto(s)

Ácidos Grasos Monoinsaturados , Liposomas , Compuestos de Amonio Cuaternario , ARN Mensajero/análisis , Transfección , Uridina Trifosfato/análogos & derivados , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Células HeLa , Humanos , Inmunohistoquímica , Microinyecciones , Precursores del ARN , Uridina Trifosfato/metabolismo

RESUMEN

In the present study, a CAT assay, a beta-galactosidase assay, and immunofluorescence analysis have been used to study the cellular uptake of the HIV-1 Tat protein. An anti-Tat MAb binding to an epitope comprising both the basic domain and the RGD sequence inhibits trans-activation by exogenous Tat. Two different full-length recombinant Tat proteins were used in these studies. The inhibitory MAb, however, recognized only one of the recombinant Tat proteins. Immunofluorescence analysis demonstrated that only the Tat protein recognized by the inhibitory anti-Tat MAb was taken up by COS and HeLa cells. This indicates that there are conformational differences between the two Tat proteins and that a correct folding of the epitope recognized by the anti-Tat MAb is required for cellular uptake. The recombinant Tat taken up by the cells was distributed between the nucleoli, the nucleoplasm, and along the nuclear membrane. Interactions between Tat and serum components were shown in vitro and also inhibition of trans-cellular trans-activation by fetal calf serum in tissue culture was demonstrated. The specific inhibition of the cellular uptake of Tat by an anti-Tat monoclonal antibody and the blocking of uptake by serum components implies specific binding of Tat to the cell membrane.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Membrana Celular/virología , Productos del Gen tat/metabolismo , Anticuerpos Anti-VIH/inmunología , VIH-1/metabolismo , Activación Transcripcional , Animales , Sitios de Unión , Línea Celular Transformada , Chlorocebus aethiops , Productos del Gen tat/antagonistas & inhibidores , Productos del Gen tat/inmunología , Duplicado del Terminal Largo de VIH , VIH-1/inmunología , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , beta-Galactosidasa/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype.

Asunto(s)

Núcleo Celular/metabolismo , Productos del Gen rev/metabolismo , VIH-1/metabolismo , Animales , Transporte Biológico , Western Blotting , Línea Celular , Dactinomicina/farmacología , Técnica del Anticuerpo Fluorescente , Productos del Gen rev/efectos de los fármacos , Productos del Gen rev/genética , Genes Dominantes , VIH-1/genética , Mutación , Empalme del ARN , ARN Mensajero/genética , ARN Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS.

Asunto(s)

Anticuerpos Monoclonales , Epítopos/análisis , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Chlorocebus aethiops , Cartilla de ADN , Exones , Productos del Gen tat/inmunología , Reordenamiento Génico , Genes de Inmunoglobulinas , VIH-1/genética , Células HeLa , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Riñón , Ratones , Ratones Endogámicos BALB C/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Linfocitos T , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

Asunto(s)

Productos del Gen rev/metabolismo , VIH-1/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Transporte Biológico Activo/efectos de los fármacos , Compartimento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dactinomicina/farmacología , Técnica del Anticuerpo Fluorescente , Productos del Gen rev/genética , Productos del Gen rev/inmunología , Anticuerpos Anti-VIH , VIH-1/genética , VIH-1/inmunología , Células HeLa , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Empalme del ARN , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.

Asunto(s)

Compartimento Celular , Núcleo Celular/química , Productos del Gen rev/aislamiento & purificación , VIH-1/química , Empalme del ARN , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Autoanticuerpos , Transporte Biológico , Núcleo Celular/ultraestructura , Cicloheximida/farmacología , Técnica del Anticuerpo Fluorescente , Productos del Gen rev/inmunología , Anticuerpos Anti-VIH/biosíntesis , VIH-1/inmunología , Células HeLa , Humanos , Interfase/fisiología , Datos de Secuencia Molecular , Biosíntesis de Proteínas/efectos de los fármacos , ARN Nuclear Pequeño/aislamiento & purificación , Ribonucleoproteínas Nucleares Pequeñas/aislamiento & purificación , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

The human immunodeficiency virus (HIV-1) encodes a transactivator protein, the product of the tat gene (tat), which is essential for virus replication. In this study, immunogold electron microscopy was used in a stably transfected Jurkat T-cell line that constitutively expresses HIV-1 tat protein to determine the subcellular and intranuclear distribution of tat protein. Two nucleocytoplasmic shuttle proteins C23/nucleolin and B23 and a third nucleolar antigen that was detected by monoclonal antibody MAb 1277 were also examined. In addition, spatial association of C23 and B23 with tat protein at several subcellular locations was examined in dual-labeling experiments. The results showed that tat protein was found in both the cytoplasm and nucleus but was especially prominent within the dense fibrillar and granular components of the nucleolus. There was little labeling of tat protein in the fibrillar centers where MAb 1277 antigen was localized at a comparatively high level. The subcellular and intranucleolar distribution of tat protein was virtually identical to the pattern seen with C23 and B23. Although the intranuclear distributions of C23, B23 and tat protein were very similar, C23 and tat protein were seldom spatially associated. In contrast, B23 and tat protein were frequently spatially associated in the nucleolus and in several other subcellular locations including the cytoplasm, nucleoplasm, at the nuclear envelope and plasma membrane. While a physical association was not directly demonstrated in this study, the spatial association between B23 and tat protein strongly suggest that such an association may exist.

Asunto(s)

Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Productos del Gen tat/biosíntesis , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Animales , Anticuerpos Monoclonales , Línea Celular , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Productos del Gen tat/análisis , VIH-1/genética , Humanos , Ratones , Ratones Endogámicos BALB C/inmunología , Microscopía Inmunoelectrónica , Proteínas Nucleares/análisis , Nucleofosmina , Fosfoproteínas/metabolismo , Linfocitos T , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Antibody binding to the p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.

Asunto(s)

VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/química , Ribonucleasa H/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Sitios de Unión de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Transcriptasa Inversa del VIH , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , ADN Polimerasa Dirigida por ARN/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Ribonucleasa H/inmunología

RESUMEN

Rotavirus infection in the Dar es Salaam area of Tanzania was studied in 99 hospitalized children with acute diarrhoea and 99 hospitalized non-diarrhoea referents matched for sex and age. Of the diarrhoea cases 43.4% had rotavirus in the stools as opposed to 15.2% of the referents. The high carrier rate among the referents represents a serious risk of nosocomial transmission. More referents than cases had serum IgG antibodies to rotavirus, 52.5% and 35.4%, respectively (P < 0.02), while there was no correlation with serum IgM and IgA or faecal IgA antibodies. The latex agglutination test had a sensitivity comparable to that of electron microscopy (100%) and a specificity of 93.8%. The Slidex test appeared to be superior to the Rotalex test in that it gives very few false-positive reactions. The SDS-PAGE patterns of 11 RNA segments were compatible with the presence of group A strains with considerable heterogeneity among the strains. Symptoms and signs and some environmental data were recorded. None of them was clearly associated with rotavirus infection among the diarrhoea cases. It is concluded that rotavirus is a major cause of acute infectious diarrhoea in Tanzania.

Asunto(s)

Infecciones por Rotavirus/epidemiología , Anticuerpos Antivirales/análisis , Diarrea/epidemiología , Diarrea/etiología , Electroforesis en Gel de Poliacrilamida , Heces/microbiología , Femenino , Humanos , Lactante , Pruebas de Fijación de Látex , Masculino , Prevalencia , Rotavirus/inmunología , Tanzanía/epidemiología

RESUMEN

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Asunto(s)

Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Epítopos/química , VIH-1/enzimología , Mapeo Peptídico , ADN Polimerasa Dirigida por ARN/inmunología , Ribonucleasa H/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/fisiología , Unión Competitiva , Epítopos/inmunología , Transcriptasa Inversa del VIH , VIH-1/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa