Expression and activation of human endogenous retroviruses of the W family in blood cells and astrocytes: implications for the pathogenesis of multiple sclerosis

Background Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, with demyelination and gliosis. Proposed pathogenic co-factors triggering MS pathogenesis are the Epstein Barr virus (EBV), and two elements of the W family of human endogenous retroviruses (HERV-W): MSRV, that forms free virions, and syncytin-1, the ERVWE1env protein; both retroelements have neuropathogenic properties. In the past we studied MSRV in MS patients in various temporal and clinical stages; in all cases, striking parallelisms between MS behaviour and MSRV/HERV-W presence/ load were found. By simultaneous detection of MSRV and HHV-6, we found a direct correlation between MS and MSRV presence/load, but not for HHV-6. MS brains over-express MSRVenv and syncytin-1 transcripts, with respect to controls, while EBV presence was not detected. Materials and methods Since late EBV seroconversion is a strong risk factor for MS development, we performed in vitro experiments on PBMC from MS patients and MSRV+ volunteers, as well as on U87-MG astroglioma cells, that were studied as such or were exposed to EBV or to recombinant EBV glycoprotein350 (EBVgp350), or to proinflammatory cytokines. The levels of MSRVenv and syncytin-1 mRNAs were evaluated by discriminatory real time RTPCR assays. Flow cytometry was used to evaluate the HERV-Wenv protein on the plasmamembrane, as well the PBMC subsets. Results Basal expression of MSRVenv and syncytin-1 occurs in astrocytes and in NK, B and monocyte cells, but not in T cells. This uneven expression is amplified in naive MS patients. Astrocyte infection by EBV and exposure to EBVgp350 stimulate the expression of HERV-W/MSRV/ syncytin-1, with requirement of the NF-kB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 are 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 The study demonstrates that there are interactions among the above proposed MS-cofactors. In vivo, a pathogenic outcome would depend on activation in abnormal situations/tissues, as it may occur in delayed EBV infection, or in the presence of particular host genetic backgrounds, or both.</br

Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for Multiple Sclerosis

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-kB 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

Multiple sclerosis and HERV-W/MSRV: A multicentric study

We designed a large multicentric study to analyse the presence of MSRV particles in blood and CSF of a large cohort of patients and controls from different European areas. 149 MS patients and 153 neurological and healthy controls were selected from Sardinia, Spain, Northern-Italy and Sweden. To avoid biological and inter-assay variability MSRV was detected within a single laboratory through nested and real-time PCR assays specific for pol and env genes. MSRV detection in blood and CSF of MS patients and controls in populations of different ethnicity gave significant differences (p<0.05 compared to neurological controls and <0.001 compared to healthy controls). The presence and viral load of MSRV are significantly associated with MS as compared to neurological and healthy controls in all ethnic groups

The Multiple sclerosis-associated retrovirus and its HERV-W endogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease

This mini-review summarizes current knowledge of MSRV (multiple sclerosis-associated retrovirus), founder member of the type W family of human endogenous retroviruses (HERVs), its pathogenic potential and association with diseases. As retrotransposable elements, HERVs behave differently from stable genes, and cannot be studied with "Mendelian genetics" concepts only. They also display complex interactions with other HERV families, and with classical viruses. These concepts may contribute to unravelling the etiopathogenesis of complex diseases such as multiple sclerosis, schizophrenia, and other chronic multifactorial diseases

Expression of HERV Genes as Possible Biomarker and Target in Neurodegenerative Diseases

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

Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis

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-&kappa;B), 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

Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells

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