RESUMEN
The HIV-1 gene promoter is a bi-directional promoter of transcription. We report the characterization of the negative sense promoter (NSP) by analysis of the effect on negative sense transcription of a series of LTR U3 region substitution mutants. Mutations in the region nt -58 to -183 (positive sense transcription initiation nt +1) reduced transcription to <15% of wild type NSP activity. This region, essential for NSP activity, was designated the core basal promoter. Over expression of NF-kappaB RelA(p65) and LEF-1 increased negative sense expression, as did over expression of H-ras oncogene, consistent with the presence of cognate sequence motifs for NF-kappaB, LEF-1 and RBF. We were also able to confirm that the NSP is a TATA-less promoter inhibited by HIV-1 Tat. Based on our findings, we propose a model for the interaction between the NSP and PSP, and the role of Tat in regulating the interaction.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/genética , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Análisis Mutacional de ADN , Regulación hacia Abajo , Productos del Gen tat/fisiología , Duplicado del Terminal Largo de VIH/genética , Humanos , Células Jurkat , Mutación/fisiología , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVE: To determine whether HIV-1 can be recovered from blood monocytes as well as resting, memory CD4 T lymphocytes of patients on highly active antiretroviral therapy (HAART) with undetectable plasma viraemia and whether infection is active or latent. DESIGN: Five patients with plasma HIV-1-RNA levels of less than 500 copies/ml for at least 3 months and less than 50 copies/ml at the time of sampling were initially selected, followed by an additional five patients with viral loads of less than 50 copies/ml for 3 months or more. METHODS: Monocytes were isolated from blood by plastic adherence, then further purified by a second adherence step or CD3 depletion before co-culture with CD8-depleted donor peripheral blood mononuclear cells. Virus isolates were examined for mutations conferring resistance to reverse transcriptase or protease inhibitors and for genotype. The highly purified monocytes were also analysed for the presence of proviral and unintegrated viral DNA and multiply spliced (MS) viral mRNA by polymerase chain reaction. RESULTS: Virus was recovered from monocytes of five patients. Sequencing of the recovered viruses did not reveal multiple drug resistance, and was consistent with a non-syncytium-inducing/CCR5 phenotype. Proviral DNA was detectable in monocytes from all subjects, and unintegrated HIV-1 DNA and MS RNA was found in four out of five populations examined. CONCLUSION: Recovery of replication-competent virus from some HAART patients indicates that monocytes can also harbour HIV-1. Detection of circular, viral DNA and spliced RNA, albeit at very low levels, in these cells suggests that their infection is recent and transcriptionally active rather than latent.
Asunto(s)
Infecciones por VIH/virología , VIH-1 , Monocitos/virología , Replicación Viral , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , VIH-1/fisiología , Humanos , Integración Viral , Latencia del VirusRESUMEN
During highly active antiretroviral therapy (HAART), HIV-1 can still persist in circulating, resting CD4+ T lymphocytes, lymph node mononuclear cells, and seminal cells of patients despite sustained suppression of plasma viremia to undetectable levels. Sanctuary sites where antiretroviral drug penetration is not optimal may allow local HIV-1 infection of cells within and passing through these tissues. Factors such as imperfect drug adherence due to complicated drug regimens may also result in tissue compartments with suboptimal drug concentrations allowing viral replication. We have examined blood monocytes from HIV-1-infected subjects being effectively treated with HAART to determine virus carriage in these cells. Monocytes were purified from peripheral blood of patients with plasma HIV-1 RNA below 50 copies/mL and who had maintained levels of plasma RNA below detection for 3 months or more. Replication-competent virus could be recovered from the majority of monocyte populations by co-culture with CD8-depleted, PHA-activated, peripheral blood mononuclear cells. Sequencing of the reverse transcriptase and protease genes of the recovered viruses did not reveal resistance to both reverse transcriptase and protease inhibitors. Continued new infection of this transitory, circulating population of cells even during prolonged, effective HAART most likely reflects ongoing, low-level HIV-1 replication within cellular reservoirs and sanctuary sites in the body.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Monocitos/virología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , Humanos , Macrófagos/virologíaRESUMEN
BACKGROUND: Previous studies of the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on HIV-1 replication in macrophages have had inconsistent results, variously reporting no effect, augmentation or inhibition of viral replication. OBJECTIVE: To investigate the regulation of HIV-1 in monocyte-derived macrophages (MDM) by GM-CSF in vitro. METHODS: The role of GM-CSF on HIV-1 replication was assessed as supernatant and intracellular p24 antigen concentrations and by HIV-1 DNA and mRNA production under different culture conditions. Expression of CD4 and CCR5 receptors was examined. The effect of GM-CSF with an E21R mutation, which binds only to the alpha-chain of GM-CSF receptor, was used as an additional control. RESULTS: GM-CSF consistently suppressed HIV-1 replication in human MDM in vitro, as assessed by supernatant and intracellular p24 antigen concentrations and HIV-1 gag mRNA expression. The inhibitory effect of GM-CSF on HIV-1 replication was observed regardless of HIV-1 strain, source of GM-CSF, stage of MDM maturation or timing of GM-CSF exposure in relation to HIV-1 infection. The effect was dose dependent and reversed by addition of a neutralizing monoclonal antibody (4D4). Flow cytometric analysis of surface expression of CD4 and CCR5 indicates that GM-CSF does not affect HIV-1 entry into MDM. Analysis of intracellular HIV-1 DNA and mRNA suggests that HIV-1 replication is inhibited at or before transcription. E21R GM-CSF had no effect on HIV-1 replication in MDM. CONCLUSIONS: GM-CSF regulates HIV-1 replication in MDM, inhibiting HIV-1 replication through binding to the beta-chain of the GM-CSF receptor.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , VIH-1/efectos de los fármacos , Macrófagos/virología , Monocitos/virología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Genes gag , Factor Estimulante de Colonias de Granulocitos/inmunología , Factor Estimulante de Colonias de Granulocitos/fisiología , Proteína p24 del Núcleo del VIH , VIH-1/fisiología , Técnicas In Vitro , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , ARN MensajeroRESUMEN
The clinical utility of a flow cytometric assay (FCA) for intracellular HIV p24 antigen was evaluated in a group of HIV-1-infected subjects. A previously described method, p24-FCA (1), was modified to minimize nonspecific staining and to include irrelevant isotype-matched control antibodies. Blood mononuclear cells from 32 HIV-1 seropositive subjects and 14 HIV-1 seronegative controls were examined. In HIV-1 seropositive individuals, the proportion of CD4+ lymphocytes that bound the p24 monoclonal and the isotype control antibodies were not different. The frequency of cells binding p24 antibodies increased with declining CD4 counts and was highest in patients with low CD4+ lymphocyte counts. Although results of p24-FCA do reflect disease progression, the high nonspecific binding of monoclonal antibodies in infected subjects obscures specific p24 binding and precludes its use as an accurate measure of infection within single cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Especificidad de Anticuerpos , Infecciones por VIH/sangre , Humanos , Isotipos de Inmunoglobulinas , Líquido IntracelularRESUMEN
OBJECTIVE: To determine the relative contribution of alveolar macrophages, peripheral blood monocytes (PBM) and peripheral blood lymphocytes (PBL) from HIV-infected individuals to HIV-1 viral load. METHODS: Alveolar macrophages were obtained by flexible bronchoscopy, and PBM and PBL by venipuncture from HIV-1-infected individuals. Alveolar macrophages and PBM were purified using immunomagnetic bead selection to deplete CD3+ and CD19+ cells from bronchoalveolar lavage and peripheral blood mononuclear cells, respectively. DNA and mRNA were extracted and gag copy number quantified using polymerase chain reaction (PCR) and reverse transcriptase PCR. The titres of infectious cell-associated HIV-1 in cells were determined by the endpoint dilution coculture technique for alveolar macrophages and PBM. RESULTS: Alveolar macrophages and PBM from HIV-1-infected subjects (n=11) contained equivalent concentrations of HIV-1 DNA and HIV-1 mRNA as determined by PCR and reverse transcriptase PCR, respectively. Antiretroviral therapy was associated with reduced viral DNA concentrations in alveolar macrophages but not in PBM. PBL had a significantly higher level of proviral DNA and mRNA than alveolar macrophages or PBM. CONCLUSIONS: Although alveolar macrophages infected in vitro are more permissive for HIV-1 replication than PBM, this difference could not be demonstrated in vivo.
Asunto(s)
ADN Viral , Genes gag , Infecciones por VIH/sangre , Infecciones por VIH/virología , VIH-1/genética , Macrófagos Alveolares/virología , Monocitos/virología , ARN Viral , Células Cultivadas , Humanos , Pulmón/patología , Pulmón/virología , Masculino , ARN Mensajero , Carga ViralAsunto(s)
Leucocitos Mononucleares/enzimología , Isomerasa de Peptidilprolil/genética , Reacción en Cadena de la Polimerasa/métodos , Seudogenes/genética , ARN Mensajero/análisis , Cartilla de ADN/genética , Desoxirribonucleasas/metabolismo , Electroforesis en Gel de Agar , Humanos , ARN/aislamiento & purificación , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
The CD4 glycoprotein is the major cellular receptor for HIV. CD4 surface expression of monocytes decreases with time in culture while their susceptibility to HIV-1 increases. Our aim was to investigate whether this phenomenon occurs in macrophages that have differentiated in vivo by investigating CD4 expression and HIV-1 infection of human alveolar macrophages (AMs). Using flow cytometry to detect CD4 expression by Leu-3a labeled indirectly with fluorescein isothiocyanate or allophycocyanin, we found that CD4 was expressed at low but detectable levels, despite the high background autofluorescence well described in AMs. This finding was supported by the detection of CD4 mRNA in AMs using RT-PCR. T cell contamination of mRNA extracts of AMs was excluded by amplifying in parallel with primers to the constant region of the T cell receptor. Despite this low level of surface CD4, recombinant soluble CD4 and anti-CD4 antibody completely inhibited HIV-1 infection of AMs. We conclude that CD4, although expressed at low levels on the surface of AMs, appears to be critical to HIV-1 infection of these cells.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Antígenos CD4/inmunología , VIH-1 , Macrófagos Alveolares/virología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citometría de Flujo , Humanos , Macrófagos Alveolares/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
Peripheral blood monocytes are resistant to productive human immunodeficiency virus type 1 (HIV-1) infection in vitro immediately after isolation. No viral cDNA (either early or late transcripts) was detected by PCR in monocytes exposed to virus on the day of isolation. In contrast, in monocytes cultured for as little as 1 day, initiated and completed reverse transcripts were readily detectable within 24 h of infection with both HIV-1(Ba-L) and primary isolates. The levels of initiated, partially completed, and completed viral DNA copies found 24 h after infection increased progressively with time in culture before infection. Unlike quiescent T lymphocytes, there appeared to be no block or delay in the integration of viral DNA into the genome of susceptible cultured monocytes. With an Alu-PCR method designed to specifically detect proviral DNA being used, integration events were found within 24 h of infection in monocytes cultured for a day or more after isolation. No integration signal was found in freshly isolated monocytes up to 7 days following exposure to the virus. Cloning and sequencing of Alu-PCR-amplified DNA confirmed integration in HIV-1-infected cultured monocytes. Our finding that in vitro replication of HIV-1 is clearly blocked prior to the initiation of reverse transcription in freshly isolated peripheral blood monocytes suggests that these cells may not be susceptible to infection in vivo. Further studies to clarify this possibility and the nature of the block to infection should provide useful information for treatment strategies against HIV-1.
Asunto(s)
VIH-1/fisiología , Monocitos/virología , Transcripción Genética , Integración Viral , Replicación Viral , Secuencia de Bases , Células Cultivadas , Duplicado del Terminal Largo de VIH , VIH-1/genética , Humanos , Datos de Secuencia MolecularRESUMEN
A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.
Asunto(s)
Donantes de Sangre , Genes nef , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/genética , VIH-1/patogenicidad , Adulto , Anciano , Composición de Base , Secuencia de Bases , Transfusión Sanguínea , Recuento de Linfocito CD4 , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Reordenamiento Génico , Genoma Viral , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , VIH-1/fisiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Familia de Multigenes , Eliminación de Secuencia , Virulencia , Replicación ViralRESUMEN
Monocytes from HIV-seronegative persons were analyzed for CD4 expression and susceptibility to infection with HIV-1 on the day of isolation and following 1, 2, and 7 days in culture. Although surface CD4 was readily detected on freshly isolated monocytes, these cells were relatively resistant to infection. After 1 to 2 days in culture, when surface expression of CD4 had decreased over 90% to near background levels, cells became susceptible to infection with HIV-1. CD4 expression on monocytes cultured for 7 days was more than four times higher than that on freshly isolated cells, and the cultured cells were fully permissive to infection. These observations suggest that the differing susceptibility of monocytes and monocyte-derived macrophages to infection with HIV-1 is not simply proportional to the level of surface CD4 expression.
Asunto(s)
Antígenos CD4/fisiología , VIH-1/fisiología , Linfocitos/inmunología , Linfocitos/virología , Monocitos/inmunología , Monocitos/virología , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Antígenos CD4/biosíntesis , Células Cultivadas , Citometría de Flujo , Seronegatividad para VIH/inmunología , VIH-1/patogenicidad , Humanos , Cinética , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/virología , Monocitos/efectos de los fármacos , Factores de TiempoRESUMEN
Rotavirus variants resistant to neutralization were selected using monoclonal antibodies (N-MAbs) raised to VP7 of rotavirus G types 2, 3, and 6. Their neutralization resistance patterns and deduced VP7 amino acid sequences were obtained. Variants selected by two G2-specific N-MAbs from the homologous parent virus RV-5 showed single amino acid (aa) mutations in the antigenic A region. However, variants selected from reassortant virus RV-5 x SA11 (all genes from SA11 virus except that encoding VP7, which was from RV-5 virus) fell into two neutralization resistance groups. The first group showed identical mutations to the variants selected from RV-5 virus. The second group showed antigenic C region mutations, either alone or in combination with a mutation at aa 69. Variants selected from G3 parent viruses glycosylated at position 238 had a mutation at aa 96 in the A region, otherwise a C-region mutation at 211 was selected. Mutations at amino acid positions 94 or 96 were selected by monoclonal antibodies specific for each of the three serotypes. G3-specific monoclonal antibodies also selected mutations at position 148 and the new position of 264. This latter mutation resulted in substitution of aspartic acid for glycine and was located in a highly conserved and hydrophobic region of VP7. A G2-specific N-MAb selected variants with a mutation at aa 190 producing a new, utilized glycosylation site which we propose to be in new antigenic site E. The positions of mutations in antigenic variants and their antigenicity were determined by parental background genes and VP7 glycosylation.
Asunto(s)
Antígenos Virales/genética , Proteínas de la Cápside , Cápside/genética , Cápside/inmunología , Rotavirus/genética , Rotavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Variación Antigénica , Antígenos Virales/química , Secuencia de Bases , Cápside/química , Bovinos , Cartilla de ADN/genética , ADN Viral/genética , Genes Virales , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutación , Pruebas de Neutralización , Rotavirus/química , Homología de Secuencia de AminoácidoRESUMEN
Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , ADN Viral/metabolismo , VIH-1/genética , VIH-1/aislamiento & purificación , Monocitos/metabolismo , Monocitos/microbiología , Linfocitos T/metabolismo , Linfocitos T/microbiología , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/patología , Secuencia de Bases , ADN Viral/análisis , ADN Viral/genética , Humanos , Datos de Secuencia Molecular , Monocitos/química , Linfocitos T/químicaRESUMEN
Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.
Asunto(s)
VIH-1/crecimiento & desarrollo , Antígeno de Macrófago-1/biosíntesis , Macrófagos/inmunología , Monocitos/inmunología , Receptores Fc/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Humanos , Molécula 1 de Adhesión Intercelular , Macrófagos/microbiología , Monocitos/microbiologíaRESUMEN
A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.
Asunto(s)
Genes nef/inmunología , Anticuerpos Anti-VIH/sangre , Proteína gp41 de Envoltorio del VIH/inmunología , Seropositividad para VIH/inmunología , Homosexualidad , Sarcoma de Kaposi/inmunología , Seropositividad para VIH/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Sarcoma de Kaposi/complicacionesRESUMEN
Monocyte--macrophages are important target cells and reservoirs for HIV. The existing methods for the quantification of infectious virus in HIV stocks are not totally satisfactory for use with macrophage cultures. We have developed an infectious focus assay for the direct quantification of virions infectious for human peripheral blood monocyte-derived macrophages adhering to plastic microtitre plates. The combination of an HIV-1 p24-antigen-specific monoclonal antibody and a beta-galactosidase-linked second antibody resulted in a sensitive and very specific assay. With 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as substrate, the assay proved to be as sensitive as p24 antigen quantification in culture supernatants.
Asunto(s)
VIH-1/crecimiento & desarrollo , Macrófagos/microbiología , Monocitos/microbiología , Productos del Gen gag/análisis , Antígenos VIH/análisis , Proteína p24 del Núcleo del VIH , VIH-1/inmunología , Humanos , Técnicas para Inmunoenzimas , Sensibilidad y Especificidad , Proteínas del Núcleo Viral/análisis , beta-Galactosidasa/metabolismoRESUMEN
Monoclonal antibodies to glycoprotein and protein antigens of infectious laryngotracheitis virus (ILTV) were divided into five groups on the basis of their reactivity in immunofluorescence and Western blotting. Group I antibodies recognised a single band of 60 K and Group II antibodies recognised bands of 205, 160, 115, 90 and 85 k in Western blotting. In immunofluorescence both these groups of antibodies reacted with antigens located in the cytoplasm of fixed virus-infected cells and they also reacted with unfixed cells, suggesting that these antigens are on the surface of virus-infected cells. While Group I monoclonal antibodies did not react with extracts of tunicamycin-treated cells, some Group II antibodies recognised bands of decreased molecular weight compared to those present in untreated cells. The reactivity of the Group II antibodies with extracts of tunicamycin-treated cells suggested that they recognised at least three different epitopes which was confirmed by ELISA additivity assays. Monoclonal antibodies of Group III, Group IV and Group V recognised several low molecular weight proteins from 45 to 24 k. Immunofluorescence studies showed that these were nuclear and cytoplasmic antigens that were not present on the surface of virus-infected cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Herpesvirus Gallináceo 1/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Glicoproteínas/inmunología , Herpesvirus Gallináceo 1/efectos de los fármacos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Tunicamicina/farmacologíaRESUMEN
The viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of infectious laryngotracheitis virus, an avian herpesvirus, were identified by in vitro labeling using [14C]glucosamine and [14C]mannose. Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain, immunoprecipitated four major viral glycoproteins of 205, 115, 90, and 60K mol wt. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus-infected cells. Monoclonal antibodies to the immunogenic glycoproteins were produced and characterized by immunoprecipitation and Western blotting. One group of monoclonal antibodies reacted only with the 60K glycoprotein, by both techniques, while a second group reacted with the 205, 115, and 90K glycoproteins in immunoprecipitation and with additional bands of 85 and 160K in Western blotting.
Asunto(s)
Antígenos Virales/análisis , Glicoproteínas/inmunología , Herpesviridae/inmunología , Herpesvirus Gallináceo 1/inmunología , Proteínas Virales/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Precipitación Química , Técnicas de Inmunoadsorción , Peso MolecularRESUMEN
The differential distribution of two SA 11 rotaviral capsid antigens in thin sections of infected cells was examined using antibody-coated colloidal gold electron-dense particles as specific post-embedding immunocyto-chemical labels. The treatment of thin sections of conventionally fixed and embedded tissue specimens with sodium metaperiodate allowed specific localization of the antigens in tunicamycin-treated, infected CV-1 cells. Both protein antigens were investigated with specific anti-rotavirus hyper-immune sera and with specific monoclonal antibodies. These studies showed that the major outer capsid glycoprotein, gp34, of SA11 rotavirus particles was mainly located within the cisternae and along the membranes of the rough endoplasmic reticulum. The antigen of the major inner capsid protein, p42, was identified attached to enveloped virus particles, and even more obviously, on laminar crystalline structures in the nucleus and cytoplasm of the infected cells.