RESUMEN
EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.
Asunto(s)
Autofagosomas/virología , Autofagia , Herpesvirus Humano 4/fisiología , Mitocondrias/metabolismo , Monocitos/virología , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/genética , Autofagosomas/metabolismo , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Diferenciación Celular/genética , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-4/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Monocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , ARN Interferente Pequeño , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.
RESUMEN
The nuclear envelope (NE), a structural element of fundamental importance for the cell, is the first barrier that meets a virus in the early stages of viral maturation. Therefore, in order to allow the passage of nucleocapsids, viruses are known to modulate the architecture of the nuclear membrane to permit a proficient viral infection. Epstein-Barr Virus (EBV), a pathogen from Herpesvirus family, possesses two well conserved proteins, BFRF1 and BFLF2, which together form the heterodimeric nuclear egress complex (NEC) that is involved in the early steps of nuclear egress. Here we show that EBV replication causes the delocalization of emerin, a cellular LEM-domain protein normally distributed on the nuclear rim. We also demonstrate that the early lytic protein BFRF1 is responsible for emerin delocalization. Expression of BFRF1 alone or in combination with BFLF2 induces emerin hyperphosphorylation. Altogether, these results suggest a novel mechanism by which EBV exploits the cellular machinery for nucleocapsid egress.
Asunto(s)
Linfocitos B/virología , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Proteínas Virales/genética , Transporte Activo de Núcleo Celular , Animales , Linfocitos B/metabolismo , Callithrix , Línea Celular Tumoral , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Proteínas Virales/metabolismo , Virión/genética , Virión/metabolismo , Ensamble de Virus , Liberación del Virus , Replicación ViralRESUMEN
We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.
Asunto(s)
Proteína 5 Relacionada con la Autofagia/metabolismo , Autofagia , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Herpesvirus Humano 8/fisiología , Monocitos/patología , Monocitos/virología , Autofagia/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosforilación/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis.
Asunto(s)
Transdiferenciación Celular , Fibroblastos/virología , Hepacivirus/fisiología , Hepatitis C/virología , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Proteína Gli2 con Dedos de Zinc/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Hepacivirus/aislamiento & purificación , Humanos , Immunoblotting , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
High mobility group box 1 (HMGB1) is an ubiquitous protein that plays different roles in the nucleus, cytoplasm, and extracellular space. It is an important DAMP molecule that allows communication between damaged or tumor cells and the immune system. Tumor cells exploit HMGB1's ability to activate intracellular pathways that lead to cell growth and migration. Papillary thyroid cancer is a well-differentiated tumor and is often used to study relationships between cells and the inflammatory microenvironment as the latter is characterized by high levels of inflammatory cells and cytokines. Anaplastic thyroid cancer is one of the most lethal human cancers in which many microRNAs and tumor suppressor genes are deregulated. Upregulation of microRNAs 221 and 222 has been shown to induce the malignant phenotype in many human cancers via inhibition of PTEN expression. In this study we suggest that extracellular HMGB1 interaction with RAGE enhances expression of oncogenic cluster miR221/222 that in turn inhibits tumor suppressor gene PTEN in two cell lines derived from human thyroid anaplastic and papillary cancers. The newly identified pathway HMGB1/RAGE/miR221/222 may represent an effective way of tumor escape from immune surveillance that could be used to develop new therapeutic strategies against anaplastic tumors.
Asunto(s)
Proteína HMGB1/genética , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Tiroides/genética , Apoptosis/genética , Proliferación Celular/genética , Proteína HMGB1/metabolismo , Humanos , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal , Neoplasias de la Tiroides/patologíaRESUMEN
Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-µ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.
Asunto(s)
Antineoplásicos/farmacología , Autofagia , Catepsina D/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Lisosomas/efectos de los fármacos , Sulfonamidas/farmacología , Transporte Activo de Núcleo Celular , Factor Inductor de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Catepsina D/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Linfoma de Efusión Primaria , Lisosomas/enzimología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Pepstatinas/farmacología , Permeabilidad , Inhibidores de Proteasas/farmacologíaRESUMEN
The discovery of microRNA (miR) represents a novel paradigm in RNA-based regulation of gene expression and their dysregulation has become a hallmark of many a tumor. In virally associated cancers, the host-pathogen interaction could involve alteration in miR expression. Epstein-Barr virus (EBV)-encoded EBNA2 is indispensable for the capacity of the virus to transform B cells in vitro. Here, we studied how it affects cellular miRs. Extensive miR profiling of the virus-infected and EBNA2-transfected B lymphoma cells revealed that oncomiR miR-21 is positively regulated by this viral protein. Conversely, Burkitt's lymphoma (BL) cell lines infected with EBNA2 lacking P3HR1 strain did not show any increase in miR-21. EBNA2 increased phosphorylation of AKT and this was directly correlated with increased miR-21. In contrast, miR-146a was downregulated by EBNA2 in B lymphoma cells. Low miR-146a expression correlates with an elevated level of IRAK1 and type I interferon in EBNA2 transfectants. Taken together, the present data suggest that EBNA2 might contribute to EBV-induced B-cell transformation by altering miR expression and in particular by increasing oncomiR-like miR-21 and by affecting the antiviral responses of the innate immune system through downregulation of its key regulator miR-146a.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/genética , MicroARNs/fisiología , Proteínas Virales/fisiología , Northern Blotting , Western Blotting , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr/genética , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Proteínas Virales/genéticaRESUMEN
To better understand the molecular mechanisms underlying the dendritic cell (DC) defects in cancer, we analyzed which signaling pathway is implicated in the abnormal monocyte differentiation into DC determined by the presence of Primary effusion lymphoma (PEL) released factors. Our results indicate that the DC, obtained in this condition, together with phenotypic abnormalities and reduced allostimulatory function, showed hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK) molecules, in comparison to the DC differentiated in the absence of PEL-released factors. The inhibition of p38 MAPK but not of STAT3 phosphorylation, with specific inhibitors, was able to revert the effect of the PEL-released factors on the DC phenotype. This study suggests that p38 MAPK signaling pathway is an important contributor to the abnormal differentiation of DC in PEL.
Asunto(s)
Diferenciación Celular , Células Dendríticas/patología , Linfoma de Efusión Primaria/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Línea Celular , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , FN-kappa B/fisiología , Fosforilación , Factor de Transcripción STAT3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is noted for its transforming potential. Yet, it also acts as a cytostatic and growth-relenting factor in Burkitt's lymphoma (BL) cells. The underlying molecular mechanisms of the growth inhibitory property of LMP1 have remained largely unknown. In this study, we show that LMP1 negatively regulates a major oncogene, TCL1, in diffuse large B-cell lymphoma (DLBCL) and BL cells. MicroRNA (miR) profiling of LMP1 transfectants showed that among others, miR-29b, is upregulated. LMP1 diminished TCL1 by inducing miR-29b through C-terminus activation region 1 (CTAR1) and CTAR2. miR-29b locked nucleic acid (LNA) antisense oligonucleotide transfection into LMP1-expressing cells reduced miR-29b expression and consequently reconstituted TCL1, suggesting that LMP1 negatively regulates TCL1 through miR-29b upregulation. The miR-29b increase by LMP1 was due to an increase in the cluster pri-miR-29b1-a transcription, derived from human chromosome 7. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase-activating function of LMP1 is important for this effect. The ability of LMP1 to negatively regulate TCL1 through miR-29b might underlie its B-cell lymphoma growth antagonistic property. As LMP1 is also important for B-cell transformation, we suggest that the functional dichotomy of this viral protein may depend on a combination of levels of its expression, lineage and differentiation of the target cells and regulation of miRs, which then directs the outcome of the cellular response.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/metabolismo , MicroARNs/metabolismo , Oncogenes/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de la Matriz Viral/farmacología , Línea Celular Tumoral , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Humanos , Linfoma de Células B/virología , MicroARNs/farmacología , Oncogenes/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/fisiologíaRESUMEN
We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.
Asunto(s)
Herpesvirus Humano 8/química , Proteínas Virales , Linfocitos B/virología , Línea Celular , Núcleo Celular , Humanos , Membrana Nuclear , Proteínas Nucleares , Sistemas de Lectura Abierta , ARN Viral/análisis , Homología de Secuencia , Proteínas Virales/análisis , Proteínas Virales/genética , Proteínas Virales/metabolismoAsunto(s)
Epigénesis Genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/patogenicidad , Linfoma/virología , Latencia del Virus , Acetilación , Metilación de ADN , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/genética , Humanos , Linfoma/patología , Regiones Promotoras Genéticas , Recombinación Genética , Células Tumorales CultivadasRESUMEN
The human herpesvirus-8 (HHV-8) has been associated with the development of Kaposi's sarcoma. A high incidence of classic Kaposi's sarcoma has been described in Sardinia, an island West of Italy's mainland. Different seroepidemiological analyses have reported that prevalence of HHV-8 infection varies worldwide: a high HHV-8 seroprevalence has been shown in Italy. The present survey was carried out to evaluate the correlation between HHV-8 infection and classic Kaposi's sarcoma incidence in northern Sardinia. Blood samples were collected from 226 healthy donors born and resident in five different areas of North Sardinia. Seroprevalence to HHV-8 was determined searching antibodies to viral lytic proteins by immunofluorescence in sera diluted at 1:10. Classic Kaposi's sarcoma incidence data spanning a period of 23 years were examined in the areas studied. The present screening revealed that seroprevalence was 35%, within a range of 15.3-46.3% in the five areas, although it should be considered that the seroprevalence to HHV-8 can be established more accurately by the combined use of different assays. Age emerged as an important risk factor. Indeed, subjects aged > 50 years showed a higher seroprevalence to HHV-8 as compared with younger individuals. A strong direct correlation between HHV-8 prevalence and classic Kaposi's sarcoma incidence has been also observed. The wide diffusion of HHV-8 in Sardinia appears to represent an important factor in the high incidence of classic Kaposi's sarcoma reported in the island. However, additional co-factors, such as age, sex, genetic traits, or viral strain pathogenicity, are likely to play a role in the development of the disease.
Asunto(s)
Anticuerpos Antivirales/sangre , Herpesvirus Humano 8/inmunología , Sarcoma de Kaposi/epidemiología , Adulto , Femenino , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Factores de Riesgo , Sarcoma de Kaposi/sangre , Estudios SeroepidemiológicosAsunto(s)
Anticuerpos Antivirales/sangre , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/virología , Herpesvirus Humano 4/genética , Proteínas de la Membrana/inmunología , Proteínas Virales/inmunología , Neoplasias de la Mama/inmunología , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Valor Predictivo de las PruebasRESUMEN
Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/biosíntesis , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas de la Matriz Viral/biosíntesis , Latencia del Virus , Linfoma de Burkitt , Genes Virales , Herpesvirus Humano 4/fisiología , Humanos , Regiones Promotoras Genéticas , Empalme del ARN , Recombinación Genética , Células Tumorales Cultivadas , Proteínas ViralesRESUMEN
The presence and variant distribution of human herpesvirus 6 (HHV-6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV-6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV-6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti-HHV-6 antibodies in patients with brain tumors similar to that found in healthy donors.
Asunto(s)
Neoplasias Encefálicas/secundario , Encéfalo/virología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 6/aislamiento & purificación , Anticuerpos Antivirales/sangre , Antígenos Virales/análisis , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/virología , ADN Viral/análisis , Herpesvirus Humano 6/genética , Humanos , Inmunohistoquímica , Leucocitos Mononucleares/virologíaRESUMEN
A microassay was developed to detect human herpesvirus 6 (HHV-6) binding to its cellular receptor using flow cytometry. Comparable results were obtained either by using HHV-6 preparations conjugated with fluorescein isothiocyanate or by indirect immunofluorescent labeling of membrane-bound virus using as primary antibody a monoclonal antibody specific for the HHV-6 gp60/110 envelope glycoprotein. Virus attachment to the plasma membrane was specific and saturable. As expected, among cell lines of various origin, maximum binding was detected on human T-lymphoid cells (HSB-2). Papain digestion of HSB-2 cells prevented HHV-6 attachment and reduced significantly virus infection, indicating the involvement of a protein-based receptor in the attachment step. After removal of the protease, virus receptors were resynthesized and their regeneration was prevented partially by cycloheximide, an inhibitor of protein synthesis. Unexpectedly, only high concentrations (mg/ml) of soluble heparan sulfate and heparin inhibited HHV-6 binding and infection. Under the same conditions, few micrograms (per ml) of heparin suppressed completely herpes simplex type 1 (HSV-1) attachment to the same cell line. Treatment of HSB-2 cells with heparitinase and heparinase, at doses that reduced significantly HSV-1 attachment, had little effect on HHV-6 binding to the cell membrane, indicating a different requirement of heparan sulfate-containing glycosaminoglycans for the two herpesviruses. These data suggest that protein components of the cellular membrane play an essential role in HHV-6 binding and infection while heparan sulfate-glycos-aminoglycans appear to be involved only partially in virus-receptor interaction.
Asunto(s)
Glicosaminoglicanos/fisiología , Herpesvirus Humano 6/metabolismo , Linfocitos/virología , Proteínas de la Membrana/fisiología , Receptores Virales/fisiología , Animales , Metabolismo de los Hidratos de Carbono , Carbohidratos/fisiología , Línea Celular , Chlorocebus aethiops , Enzimas/metabolismo , Citometría de Flujo/métodos , Glicosaminoglicanos/metabolismo , Humanos , Linfocitos/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Virales/biosíntesis , Receptores Virales/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Células VeroRESUMEN
Very little is known about Wilms' tumor gene (WT1) expression in B cells and its importance for growth regulation and differentiation. We have investigated WT1 expression in fresh B lymphocytes and in a panel of B-cell lines of normal and malignant origin, including both Epstein-Barr virus (EBV) genome negative and EBV carrying cell lines. WT1 is constitutively activated in all lymphoblastoid cell lines (LCL) derived from EBV immortalization of lymphocytes from normal donors in vitro. These cell lines are distinguished for the presence of activated B-cell markers and an unrestricted expression of viral latent genes. In contrast, WT1 expression is abrogated in normal B lymphocytes and in all Burkitt tumor derived cell lines, irrespective of the EBV genome carrying status and their phenotype pattern. A single step RT-PCR for simultaneous detection of the four spliced transcript isoforms has been applied to confirm their expression. Analysis of variant relative proportions suggested the maintenance of a balanced expression of the isoforms in LCL, as reported in non tumorous tissues. These data, together with the evidence that the replication in vitro of lymphoblastoid cells is not affected by WT1 activation following viral immortalization, support the hypothesis that gene inactivation, in addition to disrupted alternate splicing, may play a role in growth control derangements.
Asunto(s)
Linfocitos B/patología , Linfocitos B/fisiología , Regulación Neoplásica de la Expresión Génica , Genes del Tumor de Wilms , Linfocitos B/virología , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Herpesvirus Humano 4/aislamiento & purificación , HumanosRESUMEN
Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.