RESUMEN
Natalizumab is effective against relapsing-remitting multiple sclerosis (MS) but increases the risk of progressive multifocal leukoencephalopathy (PML), which is caused by the activation of the JCV polyomavirus. SF2/ASF (splicing factor2/alternative splicing factor) is a potent cellular inhibitor of JCV replication and large T-antigen (T-Ag) expression. We reported that SF2/ASF levels in blood cells increase during the first year of natalizumab therapy and decrease thereafter, inversely related to T-Ag expression, and suggested a correlation with JCV reactivation. Here, we report SF2/ASF levels of longitudinal blood samples of two patients undergoing natalizumab therapy, who developed PML while monitored, in comparison to natalizumab-treated controls and to one-off PML samples. After 6 months of therapy, SF2/ASF levels of the two cases were reduced, instead of increased, and their overall SF2/ASF levels were lower than those from natalizumab controls. Since SF2/ASF inhibits JCV, its early reduction might have a role in subsequent PML. We are aware of the limitations of the study, but the uniqueness of serial blood samples collected before and after PML onset in natalizumab-treated patients must be stressed. If confirmed in other patients, SF2/ASF evaluation could be a new and early biomarker of natalizumab-associated PML risk, allowing an 18-24-month interval before PML onset (presently ~ 5 months), in which clinicians could evaluate other risk factors and change therapy.
Asunto(s)
Factores Inmunológicos/efectos adversos , Leucoencefalopatía Multifocal Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Natalizumab/efectos adversos , Factores de Empalme Serina-Arginina/sangre , Femenino , Humanos , Huésped Inmunocomprometido , Virus JC , Leucoencefalopatía Multifocal Progresiva/sangre , Esclerosis Múltiple Recurrente-Remitente/virología , Adulto JovenRESUMEN
The JC polyomavirus (JCV) has been repeatedly but discordantly detected in healthy colonic mucosa, adenomatous polyps, and colorectal cancer (CRC), and proposed to contribute to oncogenesis. The controversies may derive from differences in JCV targets, patient's cohorts, and methods. Studies of simultaneous detection, quantification, and characterization of JCV presence/expression in paired samples of normal/altered tissues of the same patient are lacking. Therefore, we simultaneously quantified JCV presence (DNA) and expression (mRNA and protein) of T-antigen (T-Ag), Viral Protein 1 (Vp1), and miR-J1-5p in paired normal/altered tissues of CRC or polyps, and from controls. JCV signatures were found in most samples. They increased in patients, but were higher in normal mucosa than in corresponding polyp or CRC lesions. JCV non-coding control region (NCCR) DNA rearrangements increased in CRC patients, also in normal mucosa, thus before the onset of the lesion. A new ∆98bp NCCR DNA rearrangement was detected. T-Ag levels were higher in normal mucosa than in adenoma and adenocarcinoma lesions, but decreased to levels of controls in established CRC lesions. In CRC, miR-J1-5p expression decreased with CRC progression. Vp1 expression was not detected. The data indicate a JCV link with the disease, but possible JCV contributes to oncogenesis should occur at pre-polyp stages.
Asunto(s)
Neoplasias del Colon/virología , Neoplasias Colorrectales/virología , Mucosa Intestinal/virología , Virus JC/patogenicidad , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/virología , Adenoma/metabolismo , Adenoma/patología , Adenoma/virología , Anciano , Antígenos Virales de Tumores/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , ADN Viral/genética , Progresión de la Enfermedad , Femenino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/patología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/patologíaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer death worldwide and about 20% is metastatic at diagnosis and untreatable. The anti-EGFR therapy in metastatic patients is led by the presence of KRAS-mutations in tumor tissue. KRAS-wild-type CRC patients showed a positive response rate of about 70% to cetuximab or panitumumab combined with chemotherapy. MiRNAs are promising markers in oncology and could improve our knowledge on pathogenesis and drug resistance in CRC patients. This class of molecules represents an opportunity for the development of miRNA-based strategies to overcome the ineffectiveness of anti-EGFR therapy. We performed an integrative analysis of miRNA expression profile between KRAS-mutated CRC and KRAS-wildtype CRC and paired normal colic tissue (NCT). We revealed an overexpression of miR-425-5p in KRAS-mutated CRC compared to KRAS-wild type CRC and NCT and demonstrated that miR-425-5p exerts regulatory effects on target genes involved in cellular proliferation, migration, invasion, apoptosis molecular networks. These epigenetic mechanisms could be responsible of the strong aggressiveness of KRAS-mutated CRC compared to KRAS-wildtype CRC. We proved that some miR-425-5p targeted genes are involved in EGFR tyrosine kinase inhibitor resistance pathway, suggesting that therapies based on miR-425-5p may have strong potential in targeting KRAS-driven CRC. Moreover, we demonstrated a role in the oncogenesis of miR-31-5p, miR-625-5p and miR-579 by comparing CRC versus NCT. Our results underlined that miR-425-5p might act as an oncogene to participate in the pathogenesis of KRAS-mutated CRC and contribute to increase the aggressiveness of this subcategory of CRC, controlling a complex molecular network.
Asunto(s)
Neoplasias Colorrectales/genética , Epigénesis Genética/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Apoptosis/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cetuximab/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Mutación/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Panitumumab/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Human endogenous retroviruses (HERVs) are genetic parasites, in-between genetics and environment. Few HERVs retain some coding capability. Sometimes, the host has the advantage of some HERV genes; conversely, HERVs may contribute to pathogenesis. The expression of HERVs depends on several factors, and is regulated epigenetically by stimuli such as inflammation, viral and microbial infections, etc. Increased expression of HERVs occurs in physiological and pathological conditions, in one or more body sites. Several diseases have been attributed to one or more HERVs, particularly neurological diseases. The key problem is to differentiate the expression of a HERV as cause or effect of a disease. To be used as a biomarker, a correlation between the expression of a certain HERV and the disease onset and/or behavior must be found. The greater challenge is to establish a pathogenic role. The criteria defining causal connections between HERVs and diseases include the development of animal models, and disease modulation in humans, by anti-HERV therapeutic antibody. So far, statistically significant correlations between HERVs and diseases have been achieved for HERV-W and multiple sclerosis; disease reproduction in transgenic animals was achieved for HERV-W and multiple sclerosis, and for HERV-K and amyotrophic lateral sclerosis. Clinical trials for both diseases are in progress.
Asunto(s)
Biomarcadores , Retrovirus Endógenos/genética , Expresión Génica , Enfermedades Neurodegenerativas/etiología , Regulación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , PronósticoRESUMEN
The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Retrovirus Endógenos/genética , Neoplasias/virología , Oncogenes/genética , Proteínas del Envoltorio Viral/genética , Esclerosis Amiotrófica Lateral/terapia , Sistemas CRISPR-Cas , Carcinogénesis/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Retrovirus Endógenos/patogenicidad , Edición Génica , Regulación de la Expresión Génica , Humanos , Masculino , FN-kappa B/genética , Neoplasias de la Próstata , Proto-Oncogenes Mas , ARN Viral , Factores de Empalme Serina-Arginina/genética , Staphylococcus aureus/enzimologíaRESUMEN
The causes of multiple sclerosis and amyotrophic lateral sclerosis have long remained elusive. A new category of pathogenic components, normally dormant within human genomes, has been identified: human endogenous retroviruses (HERVs). These represent â¼8% of the human genome, and environmental factors have reproducibly been shown to trigger their expression. The resulting production of envelope (Env) proteins from HERV-W and HERV-K appears to engage pathophysiological pathways leading to the pathognomonic features of MS and ALS, respectively. Pathogenic HERV elements may thus provide a missing link in understanding these complex diseases. Moreover, their neutralization may represent a promising strategy to establish novel and more powerful therapeutic approaches.
Asunto(s)
Retrovirus Endógenos/genética , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/virología , Animales , Productos del Gen env/genética , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Enfermedades del Sistema Nervioso/genéticaRESUMEN
Two human endogenous retroviruses of the HERV-W family are proposed as multiple sclerosis (MS) co-factors: MS-associated retrovirus (MSRV) and ERVWE1, whose env proteins showed several potentially neuropathogenic features, in vitro and in animal models. Phase II clinical trials against HERV-Wenv are ongoing. HERV-W/MSRV was repeatedly found in MS patients, in striking parallel with MS stages, active/remission phases, and therapy outcome. The HERV-Wenv protein is highly expressed in active MS plaques. Early MSRV presence in spinal fluids predicted worst MS progression 10 years in advance. Effective anti-MS therapies strongly reduced MSRV/Syncytin-1/HERV-W expression. The Epstein-Barr virus (EBV) activates HERV-W/MSRV in vitro and in vivo, in patients with infectious mononucleosis and controls with high anti-EBNA1-IgG titers. Thus, the two main EBV/MS links (infectious mononucleosis and high anti-EBNA1-IgG titers) are paralleled by activation of HERV-W/MSRV. It is hypothesized that EBV may act as initial trigger of future MS, years later, by activating MSRV, which would act as direct neuropathogenic effector, before and during MS.
Asunto(s)
Retrovirus Endógenos , Infecciones por Virus de Epstein-Barr , Esclerosis Múltiple/virología , Herpesvirus Humano 4 , HumanosRESUMEN
Human astrocyte cells were exposed to HIV-Tat and/or epidermal growth factor (EGF), to monitor the expression of the neuropathogenic MSRV and Syncytin-1 elements of the HERV-W family of endogenous retroviruses and of TNFα. The results indicate that EGF counteracts Tat regulation of HERV-W/MSRVenv/Syncytin-1, throughout EGFR activation; this effect occurs by interfering with the induction of TNFα production by Tat. The novel effect of EGF counteraction of Tat-mediated regulation of the neuropathogenic HERV-Ws could be neuro-protective, but its actual role in the brain remains to be elucidated.
Asunto(s)
Astrocitos/virología , Retrovirus Endógenos/genética , Factor de Crecimiento Epidérmico/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Anticuerpos Monoclonales/farmacología , Astrocitos/efectos de los fármacos , Línea Celular , Retrovirus Endógenos/crecimiento & desarrollo , Retrovirus Endógenos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feto , Productos del Gen env/genética , Productos del Gen env/metabolismo , Anticuerpos Anti-VIH/farmacología , Humanos , Lipopolisacáridos/farmacología , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidoresRESUMEN
Natalizumab is effective against multiple sclerosis (MS), but is associated with progressive multifocal leukoencephalopathy (PML), fatal disease caused by the JCV polyomavirus. The SF2/ASF (splicing factor2/alternative splicing factor) inhibits JCV in glial cells. We wondered about SF2/ASF modulation in the blood of natalizumab-treated patients and if this could influence JCV reactivation. Therefore, we performed a longitudinal study of MS patients under natalizumab, in comparison to patients under fingolimod and to healthy blood donors. Blood samples were collected at time intervals. The expression of SF2/ASF and the presence and expression of JCV in PBMC were analyzed. A bell-shaped regulation of SF2/ASF was observed in patients treated with natalizumab, increased in the first year of therapy, and reduced in the second one, while slightly changed, if any, in patients under fingolimod. Notably, SF2/ASF was up-regulated, during the first year, only in JCV DNA-positive patients, or with high anti-JCV antibody response; the expression of the JCV T-Ag protein in circulating B cells was inversely related to SF2/ASF protein expression. The SF2/ASF reduction, parallel to JCV activation, during the second year of therapy with natalizumab, but not with fingolimod, may help explain the increased risk of PML after the second year of treatment with natalizumab, but not with fingolimod. We propose that SF2/ASF has a protective role against JCV reactivation in MS patients. This study suggests new markers of disease behavior and, possibly, help in re-evaluations of therapy protocols.
Asunto(s)
Interacciones Huésped-Patógeno , Factores Inmunológicos/uso terapéutico , Leucoencefalopatía Multifocal Progresiva/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Natalizumab/uso terapéutico , Factores de Empalme Serina-Arginina/genética , Adulto , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta a Droga , Femenino , Clorhidrato de Fingolimod/uso terapéutico , Regulación de la Expresión Génica , Humanos , Virus JC/efectos de los fármacos , Virus JC/genética , Virus JC/crecimiento & desarrollo , Leucoencefalopatía Multifocal Progresiva/inmunología , Leucoencefalopatía Multifocal Progresiva/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/virología , Neuroglía/efectos de los fármacos , Neuroglía/inmunología , Neuroglía/virología , Factores de Empalme Serina-Arginina/inmunología , Transducción de Señal , Activación Viral/efectos de los fármacosRESUMEN
The human genome contains remnants of ancestral retroviruses now endogenously transmitted, called human endogenous retroviruses (HERVs). HERVs can be variably expressed, and both beneficial and detrimental effects have described. This review focuses on the MSRV and syncytin-1 HERV-W elements in relationship to neurodegeneration in view of their neuro-pathogenic and immune-pathogenic properties. Multiple sclerosis (MS) and a neurodegenerative disease (neuroAIDS) are reported in this review. In vivo studies in patients and controls for molecular epidemiology and follow-up studies are reviewed, along with in vitro cellular studies of the effects of treatments and of molecular mechanisms. HERV-W/MSRV has been repeatedly found in MS patients (in blood, spinal fluid, and brain samples), and MRSV presence/load strikingly parallels MS stages and active/remission phases, as well as therapy outcome. The DNA of MS patients has increased MSRVenv copies, while syncytin-1 copies are unchanged in controls. Presence of MSRV in the spinal fluid predicted the worst MS progression, ten years in advance. The Epstein-Barr virus (EBV) activates HERV-W/MSRV both in vitro and in vivo. With respect to neuroAIDS, the HIV transactivator of transcription (Tat) protein activates HERV-W/MSRV in monocytes/macrophages and astrocytes indirectly by interaction with TLR4 and induction of TNFa. HERV-W/MSRV can be considered a biomarker for MS behavior and therapy outcome. Regarding MS pathogenesis, we postulate the possibility for EBV of an initial trigger of future MS, years later, and for MSRV of a direct role of effector of neuropathogenesis during MS. Additionally, HERV-W/MSR/syncytin-1 activation by HIV Tat could contribute to the HIV-related neurodegeneration.
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Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidad , Esclerosis Múltiple/virología , Enfermedades Neurodegenerativas/virología , Humanos , Epidemiología Molecular , Esclerosis Múltiple/epidemiología , Enfermedades Neurodegenerativas/epidemiologíaRESUMEN
Interferons (IFN) are key cytokines with multifaceted antiviral and cell-modulatory properties. Three distinct types of IFN are recognized (I-III) based on structural features, receptor usage, cellular source and biological activities. The action of IFNs is mediated by a complex, partially overlapping, transcriptional program initiated by the interaction with specific receptors. Genetic diversity, with polymorphisms and mutations, can modulate the extent of IFN responses and the susceptibility to infections. Almost all viruses developed mechanisms to subvert the IFN response, involving both IFN induction and effector mechanisms. Interactions between IFN types may occur, for both antiviral and cell-modulatory effects, in a complex interplay, involving both synergistic and antagonistic effects. Interferon-associated diseases, not related to virus infections may occur, some of them frequently observed in IFN-treated patients. On the whole, IFNs are pleiotropic biologic response modifiers, that, upon activation of thousands genes, induce a broad spectrum of activities, regulating cell cycle, differentiation, plasma membrane molecules, release of mediators, etc., that can be relevant for cell proliferation, innate and adaptive immunity, hematopoiesis, angiogenesis and other body functions.
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Interferón Tipo I/fisiología , Interferones/fisiología , Virosis/inmunología , Antivirales/inmunología , Diferenciación Celular , Proliferación Celular , Variación Genética , Humanos , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón gamma/fisiología , Interferones/genética , Interferones/inmunología , Receptor de Interferón alfa y beta/fisiología , Transducción de Señal , Activación Transcripcional , Virosis/virologíaRESUMEN
The First International Scientific Conference on Human Endogenous Retroviruses (HERVs) and Disease, Lyon-France, May 26-27th 2015, brought together scientific and medical specialists from around the world investigating the involvement of human endogenous retroviruses (HERVs) in complex human diseases.
RESUMEN
OBJECTIVE: The objective of this study is to verify whether HIV activates two endogenous retroviruses of the human endogenous retrovirus (HERV)-W family, multiple sclerosis-associated retrovirus (MSRV) and Syncytin-1, whose neuropathogenic and immunopathogenic properties could contribute to HIV-related neurodegeneration. DESIGN AND METHODS: Peripheral blood mononuclear cells, monocyte-macrophages and astrocytes were either infected by HIV or exposed to HIV-Tat, and/or other treatments. The expression of transcripts and proteins of interest was evaluated by real-time RT-PCR and western blotting assays, respectively. RESULTS: HIV and Tat increase the levels of MSRVenv mRNAs and HERV-Wenv proteins in astrocytes and in blood cells. In monocyte-macrophages, Tat also induces high levels of CCR2, CD16 and Toll-like receptor 4 (TLR4) molecules. Syncytin-1 response to Tat depends on the cell context: in monocytes, Tat stimulates MSRVenv and inhibits Syncytin-1, while in differentiated macrophages, it stimulates both elements. In primary astrocytes, Tat stimulates MSRV and Syncytin-1 indirectly, through interaction with TLR4 and induction of tumour necrosis factor-alpha (TNFα), without internalization. CONCLUSION: In-vivo consequence of the study could be that, through increase of CD16 and CCR2, Tat promotes neuroinvasion not only by HIV-infected monocytes/macrophages but also by the HERV-Ws, with their neuropathogenic potential. Also, the novel finding of TLR4 stimulation by Tat may be of relevance, as TLR4 is critical in neuroinflammation. Within central nervous system (CNS), Tat-induced TNFα could induce high levels of the HERV-Ws, in both macrophages and astrocytes, also without HIV replication. The indirect mechanism by which Tat activates the HERV-Ws through induction of TNFα could add a new piece to the puzzle of CNS pathogenesis, that is the HERV-Wenv contribute to the HIV-related neurodegeneration.
Asunto(s)
Astrocitos/virología , Retrovirus Endógenos/genética , Productos del Gen env/genética , Leucocitos Mononucleares/virología , Proteínas Gestacionales/genética , Receptor Toll-Like 4/metabolismo , Transcripción Genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Complejo SIDA Demencia/patología , Células Cultivadas , VIH/crecimiento & desarrollo , HumanosRESUMEN
BACKGROUND: Several viruses were reported as co-factors triggering the pathogenesis of multiple sclerosis (MS), including the endogenous retroviruses of the HERV-W family, that were also proposed as biomarkers of disease progression and therapy outcome. OBJECTIVE: The objective of this article is to clarify whether in MS patients treatment with natalizumab has effects on MSRV/syncytin-1/HERV-W expression and the possible relationship with disease outcome. METHODS: Peripheral blood mononuclear cells were collected from 22 patients with relapsing-remitting disease, at entry and after three, six and 12 months of treatment with natalizumab. The cell subpopulations and the expression of MSRVenv/syncytin-1/HERV-Wenv were analyzed by flow cytometry and by discriminatory env-specific RT-PCR assays. RESULTS: By flow cytometry the relative amounts of T, NK and monocyte subpopulations were shown to remain fairly constant. A relative increase of B lymphocytes was observed at three to six months (p = 0.033). The MSRVenv and syncitin-1 transcripts were reduced at six to 12 months of therapy (p = 0.0001). Accordingly, at month 12, the plasma-membrane levels of the HERV-Wenv protein were reduced (p = 0.0001). B cells, NK and monocytes but not T cells expressed the HERV-Wenv protein. None of the patients relapsed during therapy. CONCLUSION: Effective therapy with natalizumab downregulates MSRV/syncytin-1/HERV-W expression.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Retrovirus Endógenos/efectos de los fármacos , Productos del Gen env/análisis , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/virología , Proteínas Gestacionales/análisis , Adulto , Estudios de Cohortes , Femenino , Citometría de Flujo , Humanos , Leucocitos Mononucleares/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Natalizumab , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
The etiology of multiple sclerosis (MS) is still unclear. The immuno-pathogenic phenomena leading to neurodegeneration are thought to be triggered by environmental (viral?) factors operating on predisposing genetic backgrounds. Among the proposed co-factors are the Epstein Barr virus (EBV), and the potentially neuropathogenic HERV-W/MSRV/Syncytin-1 endogenous retroviruses. The ascertained links between EBV and MS are history of late primary infection, possibly leading to infectious mononucleosis (IM), and high titers of pre-onset IgG against EBV nuclear antigens (anti-EBNA IgG). During MS, there is no evidence of MS-specific EBV expression, while a continuous expression of HERV-Ws occurs, paralleling disease behaviour. We found repeatedly extracellular HERV-W/MSRV and MSRV-specific mRNA sequences in MS patients (in blood, spinal fluid, and brain samples), and MRSV presence/load strikingly paralleled MS stages and active/remission phases. Aim of the study was to verify whether HERV-W might be activated in vivo, in hospitalized young adults with IM symptoms, that were analyzed with respect to expression of HERV-W/MSRV transcripts and proteins. Healthy controls were either EBV-negative or latently EBV-infected with/without high titers of anti-EBNA-1 IgG. The results show that activation of HERV-W/MSRV occurs in blood mononuclear cells of IM patients (2Log10 increase of MSRV-type env mRNA accumulation with respect to EBV-negative controls). When healthy controls are stratified for previous EBV infection (high and low, or no anti-EBNA-1 IgG titers), a direct correlation occurs with MSRV mRNA accumulation. Flow cytometry data show increased percentages of cells exposing surface HERV-Wenv protein, that occur differently in specific cell subsets, and in acute disease and past infection. Thus, the data indicate that the two main links between EBV and MS (IM and high anti-EBNA-1-IgG titers) are paralleled by activation of the potentially neuropathogenic HERV-W/MSRV. These novel findings suggest HERV-W/MSRV activation as the missing link between EBV and MS, and may open new avenues of intervention.
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Retrovirus Endógenos/fisiología , Herpesvirus Humano 4/fisiología , Mononucleosis Infecciosa/complicaciones , Esclerosis Múltiple/virología , Activación Viral , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Mononucleosis Infecciosa/virología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Masculino , ARN Viral/genética , ARN Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Latencia del Virus , Adulto JovenRESUMEN
BACKGROUND: Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter caused by human neurotropic polyomavirus, JC virus. It is now widely accepted that pathologic strains of JCV shows unique rearrangements consist of deletions and insertions within viral NCCR. While these kinds of rearrangements are related to viral tropism and pathology of the disease, their roles in molecular regulation of JCV gene expression and replication are unclear. We have previously identified SF2/ASF as a negative regulator of JCV gene expression in glial cells. This negative impact of SF2/ASF was dependent on its ability to bind a specific region mapped to the tandem repeat within viral promoter. In this report, functional role of SF2/ASF binding region in viral gene expression and replication was investigated by using deletion mutants of viral regulatory sequences. RESULTS: The second 98-base-pair tandem repeat on Mad1 strain was first mutated by deletion and named Mad1-(1X98). In addition to this mutant, the CR3 region which served the binding side for SF2/ASF was also mutated and named Mad1-ΔCR3 (1X73). Both mutations were tested for SF2/ASF binding by ChIP assay. While SF2/ASF was associated with Mad1-WT and Mad1-(1X98), its interaction was completely abolished on Mad1-ΔCR3 (1X73) construct as expected. Surprisingly, reporter gene analysis of Mad1-(1X98) and Mad1-ΔCR3 (1X73) early promoter sequences showed two and three fold increase in promoter activities, respectively. The impact of "CR3" region on JCV propagation was also tested on the viral background. While replication of Mad1-(1X98) strain in glial cells was similar to Mad1-WT strain, propagation of Mad1-ΔCR3 (1X73) was less productive. Further analysis of the transcription mediated by Mad1-ΔCR3 (1X73) NCCR revealed that late gene expression was significantly affected. CONCLUSIONS: The results of this study reveal a differential role of CR3 region within JCV NCCR in expression of JCV early and late genes.
Asunto(s)
ADN Viral/metabolismo , Interacciones Huésped-Patógeno , Virus JC/genética , Neuroglía/virología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Inmunoprecipitación de Cromatina , Análisis Mutacional de ADN , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Factores de Empalme Serina-ArgininaRESUMEN
BACKGROUND: Proposed co-factors triggering the pathogenesis of multiple sclerosis (MS) are the Epstein Barr virus (EBV), and the potentially neuropathogenic MSRV (MS-associated retrovirus) and syncytin-1, of the W family of human endogenous retroviruses. METHODOLOGY/PRINCIPAL FINDINGS: In search of links, the expression of HERV-W/MSRV/syncytin-1, with/without exposure to EBV or to EBV glycoprotein350 (EBVgp350), was studied on peripheral blood mononuclear cells (PBMC) from healthy volunteers and MS patients, and on astrocytes, by discriminatory env-specific RT-PCR assays, and by flow cytometry. Basal expression of HERV-W/MSRV/syncytin-1 occurs in astrocytes and in monocytes, NK, and B, but not in T cells. This uneven expression is amplified in untreated MS patients, and dramatically reduced during therapy. In astrocytes, EBVgp350 stimulates the expression of HERV-W/MSRV/syncytin-1, with requirement of the NF-κB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines. CONCLUSIONS/SIGNIFICANCE: In vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/syncytin-1, in cells deriving from blood and brain. In vivo, pathogenic outcomes would depend on abnormal situations, as in late EBV primary infection, that is often symptomatic, or/and in the presence of particular host genetic backgrounds. In the blood, HERV-Wenv activation might induce immunopathogenic phenomena linked to its superantigenic properties. In the brain, toxic mechanisms against oligodendrocytes could be established, inducing inflammation, demyelination and axonal damage. Local stimulation by proinflammatory cytokines and other factors might activate further HERV-Ws, contributing to the neuropathogenity. In MS pathogenesis, a possible model could include EBV as initial trigger of future MS, years later, and HERV-W/MSRV/syncytin-1 as actual contributor to MS pathogenicity, in striking parallelism with disease behaviour.
Asunto(s)
Astrocitos/virología , Células Sanguíneas/virología , Retrovirus Endógenos/fisiología , Herpesvirus Humano 4/fisiología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/virología , Activación Viral , Adulto , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Secuencia de Bases , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Línea Celular Tumoral , Cromosomas Humanos/genética , Biología Computacional , Citocinas/farmacología , ADN/genética , Retrovirus Endógenos/efectos de los fármacos , Femenino , Productos del Gen env/genética , Productos del Gen env/metabolismo , Genes env , Sitios Genéticos/genética , Genoma Humano/genética , Herpesvirus Humano 4/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis Insercional/genética , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , ARN/genética , Activación Viral/efectos de los fármacos , Adulto JovenRESUMEN
The human neurotropic polyomavirus JC (JCV) induces a broad range of neural-origin tumors in experimental animals and has been repeatedly detected in several human cancers, most notably neural crest-origin tumors including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigens, as evident by results from in vitro cell culture and in vivo animal studies. Recently, we have shown that alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription and splicing of JCV genes in glial cells through direct association with a specific DNA motif within the viral promoter region. Here, we demonstrate that SF2/ASF suppresses large T antigen expression in JCV-transformed tumor cell lines, and the expression of SF2/ASF in such tumor cells thereby inhibits the transforming capacity of the viral tumor antigens. Moreover, down-regulation of SF2/ASF in viral-transformed tumor cell lines induces growth and proliferation of the tumor cells. Mapping analysis of the minimal peptide domain of SF2/ASF responsible for JCV promoter silencing and tumor suppressor activity suggests that amino acid residues 76 to 100 of SF2/ASF are functionally sufficient to suppress the growth of the tumor cells. These observations demonstrate a role for SF2/ASF in JCV-mediated cellular transformation and provide a new avenue of research to pathogenic mechanisms of JCV-induced tumors.
RESUMEN
Two components of the HERV-W family of human endogenous retroviruses are activated during multiple sclerosis (MS) and proposed immunopathogenic co-factors: MSRV (MS-associated retrovirus), and ERVWE1 (whose env protein, syncytin-1, reaches the plasma membrane). MSRVenv and syncytin-1 are closely related, and difficult to distinguish each other. The sequences of extracellular MSRVenv and of syncytin-1 available in GenBank were compared with those found in MS patients and controls of the cohort under study. With respect to syncytin-1, MSRVenv sequences have a 12-nucleotide insertion in the trans-membrane moiety. Based on this insertion, discriminatory real-time PCR assays were developed, that can amplify selectively either MSRVenv or syncytin-1. The data of MS patients and controls indicated that MSRV and ERVWE1 are both expressed in the brain of MS patients, while only MSRV is present in the blood; MSRV was released in culture by PBMCs of MSRV-producer individuals. These cells expressed the complete MSRVenv gene in the absence of syncytin-1 expression, up to the final, fully glycosylated envelope protein product, since western blot staining with anti-HERV-Wenv antibody detected two bands of the same molecular weight (73 and 61kDa) of the fully glycosylated and partially glycosylated HERV-Wenv uncleaved proteins. Beyond MSRVenv DNA copy numbers were more abundant in MS patients than in healthy humans, while syncytin-1 were unchanged. These findings reinforce the link between MSRV and MS.