RESUMEN
Virus shedding from the epithelial cells of the serous acini of salivary glands is a major source for the horizontal transmission of cytomegalovirus. These cells are, different to other tissues, exempt from CD8 T lymphocyte control. CD4 T lymphocytes are essential to terminate the productive infection. Here, we prove that T-B cooperation and the production of antibodies are not required for this process. For the infection with murine cytomegalovirus, mutant mice were used which do not produce antibodies because of a disrupted membrane exon of the immunoglobulin mu chain gene. Also, in these mice the virus clearance from salivary glands is a function of CD4 T lymphocytes. However, these mice clear the virus and establish viral latency with a kinetics that is distinguishable from normal mice. Reactivation from virus latency is the only stage at which the absence of antibodies alters the phenotype of infection. In immunoglobulin-deficient mice, virus recurrence results in higher virus titers. The adoptive serum transfer proved that antibody is the limited factor that prevents virus dissemination in the immunodeficient host.
Asunto(s)
Anticuerpos Antivirales/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Animales , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/transmisión , Femenino , Terapia de Inmunosupresión , Masculino , Ratones , Ratones Endogámicos C57BL , Recurrencia , Replicación Viral/inmunología , Esparcimiento de VirusRESUMEN
Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule Ld of a peptide derived from MCMV IE protein pp89 (Reddehase, M.J., J. B. Rothbard, and U.H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide 168YPHFMPTNL176 (del Val, M., H.-J. Schlicht, T. Ruppert, M.J. Reddehase, and U.H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Münch, M.J. Reddehase, and U.H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of beta-galactosidase to the same control and excluded antigen specificity. The Ld-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/cis-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC class I heavy chain, beta 2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos Virales/inmunología , Citomegalovirus/inmunología , Aparato de Golgi/inmunología , Proteínas Inmediatas-Precoces , Complejo Mayor de Histocompatibilidad/inmunología , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/microbiología , Transporte Biológico , Línea Celular , Citomegalovirus/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Precipitina , Procesamiento Proteico-Postraduccional , Mapeo RestrictivoRESUMEN
Infections are leading causes of increased morbidity and mortality of severe traumatic brain-injured (STBI) patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to investigate changes in frequency of leucocytes subpopulations in peripheral blood of patients with STBI during the course of intensive care treatment. Twenty patients with STBI were included in the study. Healthy age- and sex- volunteers served as control. Peripheral blood samples were taken from these patients at day 1, 4 and 7, and peripheral blood mononuclear cells (PBMC) were isolated. The percentage of T, B lymphocyte, NK and NKT cells as well as monocytes was analysed by simultaneous detection of surface antigens using fluorochrome-conjugated monoclonal antibodies. The two major subsets of T lymphocytes (CD3(+)CD56(-)CD4(+) and CD3(+)CD56(-)CD8(+)) and NK cells (CD3(-)CD56(+dim) and CD3(-)CD56(+bright)) were also analysed by flow cytometry. Extracranial infections were presented in 55% patients with STBI. At day 4, the percentage of T lymphocytes with cytotoxic phenotype significantly diminished and their numbers restored at day 7. The frequency of NKT cells showed the identical time-dependent pattern, whereas the percentage of NK cells diminished on day 4 but did not restore after 7 days. The frequency of B lymphocytes did not change significantly during the time investigated, whereas the percentage of monocytes increased immediately after the injury and gradually diminished. The decrease in cells with cytotoxic phenotype might explain high incidence of susceptibility to infection of patients with STBI.
Asunto(s)
Lesiones Encefálicas/inmunología , Subgrupos Linfocitarios/inmunología , Adulto , Anciano , Linfocitos B/inmunología , Lesiones Encefálicas/sangre , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Linfocitos T/inmunología , Adulto JovenRESUMEN
Recovery from primary cytomegalovirus (CMV) infection is associated with resolution of the productive infection without clearance of the virus genome from affected organs. The presence of latent CMV genome in multiple organs provides the molecular basis for recurrence of CMV within multiple organs, and explains the diversity in the organ manifestations of recrudescent CMV disease during states of immunodeficiency. As a part of a unifying concept of multifocal CMV latency and recurrence, previous work has demonstrated the importance of primary virus replication for the overall load of latent CMV in organs and the risk of recurrence. In the present report, the establishment of CMV latency was studied in a murine model in which the course of primary infection in the immunocompromised host after syngeneic bone marrow transplantation was modulated by a CD8+ T cell immunotherapy. The antiviral CD8+ effector cells limited virus replication in all organs and protected the recipients from lethal CMV disease, but after resolution of the productive infection virus DNA remained. Interestingly, the copy number of latent virus DNA in tissue did not quantitatively reflect the preceding virus production in the respective organ. Specifically, in contrast to the case in the lungs and the salivary glands, virus replication in the spleen was suppressed by CD8+ T cells to below the limit of detection; yet, virus DNA was also detected in the spleen during latency and accordingly, virus recurrence in the spleen could be induced. These findings demonstrate that the control of virus replication in a particular organ does not prevent the establishment of latency in that organ.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Infecciones por Citomegalovirus/fisiopatología , Citomegalovirus/fisiología , Transfusión de Linfocitos , Replicación Viral , Animales , Antígenos CD8 , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/inmunología , ADN Viral/análisis , Femenino , Terapia de Inmunosupresión , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Glándulas Salivales/microbiología , Bazo/inmunología , Bazo/microbiología , Subgrupos de Linfocitos T , Factores de Tiempo , Trasplante Isogénico , Ensayo de Placa ViralRESUMEN
The nucleotide sequence between the lytic origin and the gH gene of the murine cytomegalovirus genome (map units 0.416-0.455) was determined. Five of the 15 open reading frames identified encode polypeptides, which exhibit significant homology to polypeptides of human cytomegalovirus (HCMV) (UL69, UL71, and UL73) and to polypeptides of several other herpesviruses (dUTPase and helicase/primase). The location in the genome of the five open reading frames and their direction of transcription is perfectly conserved between murine cytomegalovirus (MCMV) and HCMV. These data suggest that MCMV and HCMV have a highly related genome organization. Thus, MCMV offers a good model for molecular and functional analysis of cytomegalovirus genes in vitro and in vivo.
Asunto(s)
Genes Virales , Muromegalovirus/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , ADN Viral , Genoma Viral , Herpesviridae/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Péptidos/metabolismoRESUMEN
Cytomegalovirus (CMV), similar to other members of the Herpesviridae family, can establish both persistent and latent infections. Each of the CMVs that are found in many animal species replicates in the salivary gland, and oral secretion represents a source of horizontal transmission. Locally restricted replication characterizes the immunocompetent individual, whereas in the immunocompromised host, protean disease manifestations occur due to virus dissemination. The virus is cleared by immune surveillance, and CD8+ T lymphocytes play a major role. Remarkably, certain cell types of salivary gland tissues are exempt from CD8+ T-lymphocyte control of murine CMV infection and require the activity of CD4+ T lymphocytes. The results presented here suggest that this activity is a function of Th1 cells. Neutralization of endogenous gamma interferon abrogated the antiviral activity of Th1 cells but not that of CD8+ T lymphocytes in other tissues. Neutralization of endogenous gamma interferon did not interfere with the induction of the cellular and humoral immune response but acted during the effector phase. Recombinant gamma interferon could not replace the function of Th1 cells in vivo and had limited direct antiviral activity in vitro. The results therefore suggest that gamma interferon represents one, but not the only, essential factor involved in salivary gland clearance, establishment of CMV latency, and, eventually, the control of horizontal transmission.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Interferón gamma/fisiología , Glándulas Salivales/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/microbiología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Pruebas de NeutralizaciónRESUMEN
Mouse cytomegalovirus (MCMV) functions expressed at the beginning of the early phase of the viral replication cycle interfere with the major histocompatibility complex (MHC) class I-restricted pathway of antigen presentation (M. J. Reddehase, M. R. Fibi, G. M. Keil, and U. H. Koszinowski, J. Virol. 60:1125-1129, 1986; M. Del Val, K. Münch, M. J. Reddehase, and U. H. Koszinowski, Cell 58:305-315, 1989). Nascent MHC class I heavy chains associate with beta 2-microglobulin and peptide, but the assembled trimolecular complex is retained in the endoplasmatic reticulum/cis-Golgi compartment (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). To locate the responsible genomic region, the cytoplasmic retention of MHC class I molecules after injection of MCMV DNA was tested. The function was mapped to the HindIII E fragment. A recombinant MCMV deletion mutant delta MS94.5 lacking 15.8 kb in HindIII-E was constructed. Restoration of MHC class I molecule maturation and recognition of antigenic peptides by cytolytic T lymphocytes during the first hours of the early phase in mutant virus-infected cells proved the correct location to a 6.8-kb region in the HindIII E fragment. At later stages of the early phase, membrane-resident MHC class I molecules and cytolytic T lymphocyte recognition disappeared in delta MS94.5 mutant virus-infected cells. These results demonstrate that more than one early-gene function of MCMV affects the MHC class I pathway of antigen presentation. The redundant MHC class I-reactive functions target the transport of MHC class I molecules at different steps.
Asunto(s)
Citomegalovirus/genética , Citomegalovirus/inmunología , Genoma Viral , Antígenos de Histocompatibilidad Clase I/fisiología , Células 3T3 , Animales , Anticuerpos Monoclonales , Ciclo Celular , División Celular , Supervivencia Celular , Células Cultivadas , ADN Viral/análisis , Embrión de Mamíferos , Fibroblastos , Técnica del Anticuerpo Fluorescente , Genes Virales , Antígenos de Histocompatibilidad Clase I/análisis , Cinética , Ratones , Ratones Endogámicos BALB C , Mapeo Restrictivo , Eliminación de SecuenciaRESUMEN
Interferon gamma (IFN gamma) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN gamma cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNF alpha) in the antiviral defence. Neutralization of endogenous TNF alpha abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFN gamma and TNF alpha. Depletion of endogenous TNF alpha in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNF alpha also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNF alpha and IFN gamma could not limit virus replication in vivo. The results suggest that, similar to IFN gamma, TNF alpha is an integral part of the protective mechanisms involved in cytomegalovirus clearance.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8 , Citomegalovirus/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/inmunología , Replicación Viral/inmunologíaRESUMEN
The effects of long acting somatostatin analog SMS 201-995 were examined in vivo on: 1) lymphoid morphostasis and functional reactivity of cells obtained from SMS treated donors, 2) on humoral, and 3) cellular type of immunity; and in vitro on: 1) blastic transformation of lymphocytes stimulated by activators of different transmembrane pathways (CD2 by PHA and CD3/TCR by anti-CD3 monoclonal antibody and by allogeneic cells) and 2) on growth and secretory activity of several hybridoma cell lines. The data have shown that SMS in vivo decreases the proportion of CD4+, CD5+ and Ig+ cells in spleen. The reactivity of these cells to Con A was suppressed, but their spontaneous blastic transformation was increased. SMS suppressed also the plaque forming cells generation and proliferation of cells in popliteal lymph nodes during the local host versus graft reaction. The former immunosuppression was abrogated with the use of growth hormone, while in the latter, the time dependent changes in spleen composition were also noticed. The data obtained in vitro revealed that SMS may inhibit only the CD2-induced blastogenesis (in early and late interval after the use of PHA). SMS inhibited also the spontaneous growth and/or secretion of antibodies in some hybridoma cell lines.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Terapia de Inmunosupresión , Somatostatina/análogos & derivados , Somatostatina/farmacología , Animales , Anticuerpos Monoclonales , Antígenos CD/efectos de los fármacos , Antígenos CD/inmunología , Movimiento Celular , Hibridomas/efectos de los fármacos , Técnicas In Vitro , Activación de Linfocitos , Ratones , Neuroinmunomodulación , Bazo/citología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon-gamma (IFN-gamma). We now demonstrate that IFN-gamma and tumour necrosis factor alpha (TNF-alpha) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-gamma and TNF-alpha reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.
Asunto(s)
Interferón gamma/farmacología , Muromegalovirus/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacos , Animales , Células Cultivadas , ADN Viral/efectos de los fármacos , Sinergismo Farmacológico , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/fisiología , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacos , Proteínas Virales/efectos de los fármacos , Virión/efectos de los fármacos , Cultivo de Virus , Replicación Viral/fisiologíaRESUMEN
The sequence of the gene encoding glycoprotein H (gH) of murine cytomegalovirus (MCMV) strain Smith was determined and compared with the sequence of the gH of MCMV strain K181. Transcriptional analysis showed that gH is encoded by a large mRNA of 5.0 kb, which is synthesized late in infection. A recombinant vaccinia virus expressing the MCMV gH open reading frame was constructed (Vac-gH). Anti-MCMV serum precipitated a protein of 87K from Vac-gH-infected cells. Reactivity with a monoclonal antibody showed the identity of the MCMV gH with a 87K envelope glycoprotein described previously by Loh and Qualtiere. Immunization of mice with the Vac-gH recombinant gave rise to an anti-gH serum, which neutralized MCMV without complement in vitro.
Asunto(s)
Glicoproteínas/biosíntesis , Muromegalovirus/genética , Virus Vaccinia/metabolismo , Proteínas Virales/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Genes Virales/genética , Glicoproteínas/genética , Glicoproteínas/inmunología , Inmunización , Ratones , Datos de Secuencia Molecular , Muromegalovirus/inmunología , Sistemas de Lectura Abierta , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Several herpesviruses, including cytomegalovirus, induce receptors for the Fc domain of murine immunoglobulin G (IgG) molecules. Viral genes coding for these receptors have been characterized only for alphaherpesviruses. In this report, we describe a new approach that led to the identification of an Fc receptor (FcR) of murine cytomegalovirus (MCMV). The Fc fragment of IgG precipitated glycoproteins (gp) of 86 to 88 and 105 kDa from MCMV-infected cells. Deglycosylation by endoglycosidase F resulted in a protein with a molecular mass of 64 kDa. Injection of complete MCMV DNA or of DNA fragments, and the subsequent testing of cytoplasmic binding of IgG by immunofluorescence microscopy, was used to search for the coding region in the MCMV genome. The gene was located in the HindIII J fragment, map units 0.838 to 0.846, where an open reading frame of 1,707 nucleotides predicts a gp of 569 amino acids with a calculated molecular mass of 65 kDa. The sequence of this gp is related to those of the gE proteins of herpes simplex virus type 1 and varicella-zoster virus. The defined length of the mRNA, 1,838 nucleotides, was in agreement with that of a 1.9-kb RNA expressed throughout the replication cycle, starting at the early stages of infection. Expression of the gene fcr1 by recombinant vaccinia virus resulted in the synthesis of gp86/88 and gp105, each with FcR properties, and the correct identification of the gene encoding the FcR was confirmed by the DNA injection method.
Asunto(s)
Citomegalovirus/genética , Genes Virales , Glicoproteínas de Membrana/biosíntesis , Receptores Fc/biosíntesis , Proteínas Virales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Citomegalovirus/inmunología , Cartilla de ADN , ADN Complementario , Embrión de Mamíferos , Vectores Genéticos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Cinética , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores Fc/aislamiento & purificación , Receptores Fc/metabolismo , Mapeo Restrictivo , Virus VacciniaRESUMEN
Although the relative contribution of different immune effector functions to clearing tissues of cytomegalovirus is controversial, the contribution of CD8+ T lymphocytes has generally been accepted as essential. In this report, we show that under certain conditions the CD8+ T-lymphocyte subset can be dispensable for clearance of cytomegalovirus. Mice depleted of the CD8+ T-lymphocyte subset eliminated infectious virus with a clearance kinetics similar to that of normal mice. Adoptive transfer studies revealed that the limitation of virus spread required the cooperation between the CD4+ subset and other cells. Comparison between protective functions generated in fully immunocompetent and in CD8- mice demonstrated that elimination of the CD8+ subset before infection altered the quality of the antiviral immune response. The compensatory protective activity gained by CD4+ cells in CD8- mice was absent in normal mice recovering from virus infection.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Inmunidad Celular , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8 , Citomegalovirus/inmunología , Citotoxicidad Inmunológica , Ratones , Ratones Endogámicos BALB C , Trasplante de Piel/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
An immediate-early protein of murine cytomegalovirus (MCMV), pp89, elicits an immunodominant and protective major histocompatibility complex (MHC) class I Ld-restricted CD8+ T-lymphocyte response. Remarkably, presentation of the naturally processed peptide of pp89, the nonapeptide YPHFMPTNL, is abolished during permissive MCMV infection in vitro. This defect in pp89 presentation is due to the expression of MCMV early gene functions that specifically block the transport of peptide-charged MHC class I complexes to the cell surface (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). Here, we demonstrate that MCMV-specific CD8+ T lymphocytes can reconstitute pp89 presentation in a parakrine fashion. The lymphocytes mediate the restoration of antigen presentation by MCMV-infected cells by releasing gamma interferon (IFN-gamma). IFN-gamma has no effect on synthesis and stability of the viral antigen pp89 nor does it interfere with the expression of viral early genes and their inhibitory effect on MHC class I molecular maturation. IFN-gamma results in a 25-fold increase in the synthesis of MHC class I molecules and a similar increase in the abundance of pp89-derived peptide. Many of the MHC molecules remain retained by the viral effect, but a surplus of MHC molecules escapes the effect and provides the effective surface presentation of the peptide. Adoptive cell transfer studies demonstrate the IFN-gamma dependence of CD8+ T-lymphocyte function in vivo. Altogether, these data reconcile the paradoxical findings of an impaired pp89 presentation in vitro in parallel with pp89-specific CD8+ T-cell protection in vivo. The results also imply a role of IFN-gamma in the T-lymphocyte-mediated control of cytomegalovirus infection. The known propensity of cytomegalovirus to cause serious disease in the immunocompromised host is discussed in the light of these findings.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/inmunología , Interferón gamma/farmacología , Secuencia de Aminoácidos , Animales , Antígenos Virales/biosíntesis , Transporte Biológico , Antígenos CD8/inmunología , Citomegalovirus/crecimiento & desarrollo , Regulación de la Expresión Génica , Genes MHC Clase I , Antígenos H-2/inmunología , Proteínas Inmediatas-Precoces/biosíntesis , Inmunidad Celular , Inmunoterapia Adoptiva , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Replicación ViralRESUMEN
Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Muromegalovirus/metabolismo , Proteínas Virales/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/inmunología , Regulación hacia Abajo , Endocitosis/genética , Inhibidores Enzimáticos/farmacología , Regulación Viral de la Expresión Génica/genética , Genes Virales/genética , Inmunohistoquímica , Leupeptinas/farmacología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/genética , Transfección/genética , Proteínas Virales/genética , Microglobulina beta-2/metabolismoRESUMEN
It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability of beta 2-microglobulin (beta 2m) and also the degree of peptide charging of the MHC class I heavy chain beta 2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in beta 2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8+ T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in beta 2m mutant mice. In addition, there was no indication of diminished virus propagation in beta 2m-/- embryonic fibroblasts. beta 2m-/- mice suffered from the lack of CD8+ T lymphocytes that was partially compensated for by the function of CD4+ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the beta 2m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Antígenos de Histocompatibilidad Clase I/fisiología , Animales , Linfocitos T CD8-positivos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratas , Microglobulina beta-2/deficienciaRESUMEN
Cytomegaloviruses (CMVs) represent prototypic viruses of the beta-subgroup of herpesviruses. Murine cytomegalovirus (MCMV) infects mice as its natural host. Among viruses, CMVs have evolved the most extensive genetic repertoire to subvert MHC class I functions. To date three MCMV proteins have been identified which affect MHC I complexes. They are encoded by members of large virus-specific gene families located at either flanking region of the 235 kb MCMV genome. The MHC I subversive genes belong to the early class of genes and code for type I transmembrane glycoproteins. The m152-encoded 37/40 kDa glycoprotein interacts with MHC I transiently and retains class I complexes in the endoplasmic reticulum (ER) Golgi intermediate compartment on its journey to the endolysosome. In contrast, the m06-encoded glycoprotein of 48 kDa complexes tightly with ternary MHC class I molecules in the FR. Due to sorting signals in its cytoplasmic tail, gp48 redirects MHC I to endolysosomal compartments for proteolytic destruction. Likewise, the 34 kDa glycoprotein encoded by m04 binds tightly to MHC class I complexes in the ER but the gp34/MHC I complex reaches the plasma membrane. The CD8+ T-cell-dependent attenuation of a m152 deletion mutant virus proves for the first time that inhibition of antigen presentation is indeed essential for the biological fitness of CMVs in vivo.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Muromegalovirus/inmunología , Animales , Proteínas Portadoras/inmunología , Retículo Endoplásmico/inmunología , Glicoproteínas/inmunología , Aparato de Golgi/inmunología , Infecciones por Herpesviridae/virología , Humanos , Ratones , Muromegalovirus/genética , Mutación , Proteínas ViralesRESUMEN
An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis. While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule. In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection. However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations. Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/prevención & control , Lipopolisacáridos/inmunología , Antígenos O/inmunología , Antígenos O/farmacología , Animales , Afinidad de Anticuerpos , Recuento de Colonia Microbiana , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Inmunoterapia , Klebsiella/inmunología , Klebsiella/patogenicidad , Hígado/microbiología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Peritonitis/inmunología , Peritonitis/microbiología , Sepsis/inmunología , Sepsis/microbiología , Organismos Libres de Patógenos Específicos , Bazo/microbiologíaRESUMEN
The principle by which mouse cytomegalovirus blocks antigen presentation in the MHC class I pathway was investigated. The responsible gene m152, encoding a type I transmembrane glycoprotein of 40 kDa, is a member of a gene family located in the right-hand terminal region of the 230 kb virus genome. Expression of m152 in murine and human cells arrested the export of mouse class I complexes from the ER-Golgi intermediate compartment/cis-Golgi compartment and inhibited lysis by cytotoxic T cells. The plasma membrane transport of human MHC class I molecules was not affected. The deletion of the cytoplasmic tail of gp40 did not lift its effect on class I molecule export, indicating that this protein differs in its functions from known immunosubversive viral gene products and represents a novel principle by which a herpesvirus shuts off MHC class I function.
Asunto(s)
Retículo Endoplásmico/metabolismo , Genes Virales , Aparato de Golgi/metabolismo , Antígenos H-2/metabolismo , Infecciones por Herpesviridae/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Muromegalovirus/inmunología , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/microbiología , Transporte Biológico , Compartimento Celular , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Hexosaminidasas/metabolismo , Humanos , Células L , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes , Relación Estructura-Actividad , Linfocitos T Citotóxicos/inmunología , Transcripción Genética , Proteínas Virales/genética , Replicación ViralRESUMEN
Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV-encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.