Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis.

OBJECTIVES: Reactivation of HERV-K(HML-2) has been found in subsets of individuals with amyotrophic lateral sclerosis (ALS). This study examines the antibody response against HML-2 in ALS and analyzes its clinical relevance. METHODS: Antibodies to HML-2 envelope (env) were analyzed using a peptide array for epitope mapping and by a peptide enzyme-linked immunosorbent assay (ELISA) in 242 healthy donors, and 243 ALS and 85 multiple sclerosis (MS) individuals. Extracellular levels of HML-2 were analyzed by digital polymerase chain reaction (PCR). RESULTS: Antibodies in the sera of ALS individuals recognized more HML-2 env peptides compared to healthy controls (p < 0.0001). ALS individuals had higher levels of HML-2 than healthy donors (p = 0.02) and higher antibody levels against a select HML-2 env peptide compared to healthy donors or individuals with multiple sclerosis (p < 0.0001). 55.14% of ALS compared to 21.16% of healthy donors and 13.10% of MS individuals had antibodies against the HML-2 peptide (AUC = 0.769, p < 0.0001). Levels of extracellular HML-2 DNA in serum (p = 0.02) and the number of HML-2 env peptides recognized by ALS sera (p = 0.02) correlated with disease duration. Among ALS individuals, lower levels of HML-2 antibodies were associated with a definite diagnosis per EL Escorial criteria (p = 0.03), and with a lower predicted (p = 0.02) and observed survival (p = 0.03). INTERPRETATION: There is a differential antibody response against specific epitopes of HML-2 env in ALS and controls, suggesting epitope spreading, likely due to persistent antigenic exposure following reactivation of the viral genes. Low levels of antibodies to HML-2 env in ALS are associated with poor prognosis and decreased survival probability. ANN NEUROL 2022;92:782-792.

Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic.

OBJECTIVE: Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env. METHODS: Env neurotoxicity was detected in ALS cerebrospinal fluid and confirmed using recombinant Env protein in a cell-based assay and a mouse model. The mechanism of neurotoxicity was assessed with immunoprecipitation followed by mass spectrometry and Western blot, and by screening a panel of inhibitors. RESULTS: We observed that recombinant HML-2 Env protein caused neurotoxicity resulting in neuronal cell death, retraction of neurites, and decreased neuronal electrical activity. Injection of the Env protein into the brains of mice also resulted in neuronal cell death. HML-2 Env protein was also found in the cerebrospinal fluid of patients with sporadic ALS. The neurotoxic properties of the Env and the cerebrospinal fluid could be rescued with the anti-Env antibody. The Env was found to bind to CD98HC complexed to ß1 integrin on the neuronal cell surface. Using a panel of compounds to screen for their ability to block Env-induced neurotoxicity, we found that several compounds were protective and are linked to the ß1 integrin pathway. INTERPRETATION: HERV-K Env is released extracellularly in ALS and causes neurotoxicity via a novel mechanism. Present results pave the way for new treatment strategies in sporadic ALS. ANN NEUROL 2022;92:545-561.

Retroviral Elements in Pathophysiology and as Therapeutic Targets for Amyotrophic Lateral Sclerosis.

The study of the role of retroviruses in amyotrophic lateral sclerosis (ALS) dates back to the 1960s shortly after transposable elements themselves were first discovered. It was quickly realized that in wild mice both horizontal and vertical transmissions of retroviral elements were key to the development of an ALS-like syndrome leading to the postulate that endogenous retroviruses (ERVs) contribute significantly to the pathogenicity of this disease. Subsequent studies identified retroviral reverse transcriptase activity in brains of individuals with ALS from Guam. However, except for a single study from the former Soviet Union, ALS could not be transmitted to rhesus macaques. The discovery of an ALS-like syndrome in human immunodeficiency virus (HIV) and human T cell leukemia virus infected individuals led to renewed interest in the field and reverse transcriptase activity was found in the blood and cerebrospinal fluid of individuals with sporadic ALS. However, exogenous retroviruses could not be found in individuals with ALS which further reinforced the possibility of involvement of a human ERV (HERV). The first demonstration of the involvement of a HERV was the discovery of the activation of human endogenous retrovirus-K subtype HML-2 in the brains of individuals with ALS. The envelope protein of HML-2 is neurotoxic and transgenic animals expressing the envelope protein develop an ALS-like syndrome. Activation of HML-2 occurs in the context of generalized transposable element activation and is not specific for ALS. Individuals with HIV-associated ALS show a remarkable response to antiretroviral therapy; however, antiretroviral trials in ALS down-regulate HML-2 without ameliorating the disease. This highlights the need for specific drugs to be developed against HML-2 as a novel therapeutic target for ALS. Other approaches might include antisense oligonucleotides, shRNA targeted against the envelope gene or antibodies that can target the extracellular envelope protein. Future clinical trials in ALS should consider combination therapies to control these ERVs.

SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Syncytin-1/HERV-W envelope is an early activation marker of leukocytes and is upregulated in multiple sclerosis patients.

Syncytin-1 is the envelope protein of the human endogenous retrovirus W (HERV-W). It has been related to multiple sclerosis (MS) but its role in cellular immunity and its pathogenic mechanism in the autoimmune context are not fully understood. We analyzed syncytin-1 levels in peripheral blood mononuclear cells (PBMC) subsets from healthy donors, MS patients in relapse or remission, and patients with acute infections by flow cytometry. PBMC cultures were also prepared to analyze protein expression kinetics. MS patients had higher levels of syncytin-1 levels than controls. We found that syncytin-1 is elevated in monocytes during MS relapses and infections. Cells expressing syncytin-1, including monocytes, T and B lymphocytes, and NKs presented mainly an activated phenotype and, upon stimulation with LPS, its levels increased rapidly on antigen-presenting cells. Syncytin-1 ligation promoted the activation of monocytes, as demonstrated by the upregulation of CD80 and the nonclassical subset CD14low CD16+ . Our results suggest an important role for syncytin-1 in the activation of leukocytes. Given that the expression of syncytin-1 is upregulated in MS patients, this protein might be contributing to the autoimmune cascade in the disease.

Human endogenous retrovirus-K (HML-2): a comprehensive review.

The human genome contains a large number of retroviral elements acquired over the process of evolution, some of which are specific to primates. However, as many of these are defective or silenced through epigenetic changes, they were historically considered "junk DNA" and their potential role in human physiology or pathological circumstances have been poorly studied. The most recently acquired, human endogenous retrovirus-K (HERV-K), has multiple copies in the human genome and some of them have complete open reading frames that are transcribed and translated, especially in early embryogenesis. Phylogenetically, HERV-K is considered a supergroup of viruses. One of the subtypes, termed HML-2, seems to be the most active and hence, it is the best studied. Aberrant expression of HML-2 in adult tissues has been associated with certain types of cancer and with neurodegenerative diseases. This review discusses the discovery of these viruses, their classification, structure, regulation and potential for replication, physiological roles, and their involvement in disease pathogenesis. Finally, it presents different therapeutic approaches being considered to target these viruses.

Study of the anti-JCV antibody levels in a Spanish multiple sclerosis cohort.

BACKGROUND: One of the risk factor to develop progressive multifocal leukoencephalopathy (PML) among natalizumab-treated patients is the presence and high levels of anti-JCV antibodies. Our purpose was to test the association of different clinical and demographic variables with the presence and levels of anti-JCV antibodies in a Spanish cohort of patients with multiple sclerosis (MS) during natalizumab treatment. MATERIALS AND METHODS: All patients with MS from two hospitals with at least one measure of the anti-JCV antibodies levels (2011-2014) were recruited, among them were two PML cases. Anti-JCV antibody levels were assessed using two-step ELISA. RESULTS: A total of 1061 patients (16·3% natalizumab-treated) participated in this study. The seropositivity rate of anti-JCV antibodies was 58·2%. It increased with age (Pcorrected = 0·00005) and was lower among HLA-DRB1*15:01 carriers (Pcorrected = 0·049). The two patients with PML were HLA-DRB1*15:01 carriers. We had at least three quarterly anti-JCV antibody measurements (index value) from 137 patients, whose levels did not increase during natalizumab treatment. However, 5·8% of these patients had an increase of the index value higher of one point in a maximum of 6 months, something that was more frequently observed (P = 0·054) among patients treated with immunosuppressant prior to natalizumab onset. CONCLUSIONS: Old age and HLA-DRB1*15:01 were the factors that influence positively and negatively, respectively, our anti-JCV antibody prevalence, although our both PML cases were HLA-DRB1*15:01carriers. Most of our patients showed a stable anti-JCV antibody index values during natalizumab treatment.

Anti-JCV antibodies detection and JCV DNA levels in PBMC, serum and urine in a cohort of Spanish Multiple Sclerosis patients treated with natalizumab.

One of the most effective multiple sclerosis (MS) treatment is natalizumab. Nevertheless, it has been associated with an increased risk of progressive multifocal leukoencephalopathy (PML) caused by the JC virus (JCV). Our main objective was to assess the utility of testing JCV-DNA, apart from anti-JCV antibodies, to determine which natalizumab-treated MS patients has been previously in contact with the virus. For this purpose, 138 MS natalizumab/non-natalizumab treated patients participated in several studies. Cross-sectional study (CS): association of several epidemiological variables with anti-JCV antibodies and JCV-DNA levels in PBMC/serum/urine. First longitudinal study (A): evaluation of JCV-DNA prevalence in urine throughout the treatment. Second longitudinal study (B): simultaneous assessment of antibodies and viral DNA levels in PBMC/serum/urine at two time points. CS: The seropositivity rate for anti-JCV antibodies (62.3 %) and JCV prevalence in urine (59.4 %) were similar; although 26 % of our population was positive only using one of the two techniques. A: The viral prevalence in urine seemed to increase between the baseline visit and the others (Baseline-Visit/V18months, p = 0.006). B: Our rate of positive antibody seroconversion was 36 %. Nearly all patients with detectable JCV-DNA levels in PBMC excreted the virus intermittently in urine; while our PML case, positive in PBMC and serum samples 2 month before the PML, excreted JCV permanently. In conclusion, the determination of JCV DNA levels in urine could be complementary to anti-JCV antibodies for identifying MS patients who has been infected by the JCV. Further research would be necessary to understand the different JCV excretion profiles in urine.

Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease.

BACKGROUND: The envelope protein from multiple sclerosis (MS) associated retroviral element (MSRV), a member of the Human Endogenous Retroviral family 'W' (HERV-W), induces dysimmunity and inflammation. OBJECTIVE: The objective of this study was to confirm and specify the association between HERV-W/MSRV envelope (Env) expression and MS. METHODS: 103 MS, 199 healthy controls (HC) and controls with other neurological diseases (28), chronic infections (30) or autoimmunity (30) were analysed with an immunoassay detecting Env in serum. Env RNA or DNA copy numbers in peripheral blood mononuclear cells (PBMC) were determined by a quantitative polymerase chain reaction (PCR). Env was detected by immunohistology in the brains of patients with MS with three specific monoclonals. RESULTS: Env antigen was detected in a serum of 73% of patients with MS with similar prevalence in all clinical forms, and not in chronic infection, systemic lupus, most other neurological diseases and healthy donors (p<0.01). Cases with chronic inflammatory demyelinating polyneuropathy (5/8) and rare HC (4/103) were positive. RNA expression in PBMC and DNA copy numbers were significantly elevated in patients with MS versus HC (p<0.001). In patients with MS, DNA copy numbers were significantly increased in chronic progressive MS (secondary progressive MS vs relapsing-remitting MS (RRMS) p<0.001; primary progressive MS vs RRMS -<0.02). Env protein was evidenced in macrophages within MS brain lesions with particular concentrations around vascular elements. CONCLUSION: The association between MS disease and the MSRV-type HERV-W element now appears quite strong, as evidenced ex-vivo from serum and PBMC with post-mortem confirmation in brain lesions. Chronic progressive MS, RRMS and clinically isolated syndrome show different ELISA (Enzyme-Linked Immunosorbent Assay) and/or PCR profiles suggestive of an increase with disease evolution, and amplicon sequencing confirms the association with particular HERV-W elements.

JC virus in cerebrospinal fluid samples of multiple sclerosis patients at the first demyelinating event.

OBJECTIVE: To evaluate the possible involvement of JC virus (JCV) in the aetiology of multiple sclerosis (MS), through the comparison of DNA prevalences and viral loads of JCV in cerebrospinal fluid (CSF) of MS patients at the first demyelinating event and subjects suffering from other neurological diseases (OND). METHODS: Seventy-three CSF samples (43 from MS patients at the first demyelinating event, and 30 from patients with OND) were collected; all MS cases were followed up from 1 to 6.7 years after they were diagnosed with clinically definite MS. DNA was extracted and analysed by real-time PCR for the detection of JCV genomes. RESULTS: We found JCV DNA in the CSF of two MS patients (4.7%) with a mean viral load of 2.1 and 6.7 copies/mL of CSF. Among the patients of the OND group we did not find any positive sample. We did not find any difference in the course of the disease between MS patients with and without JCV genomes in their CSF along the follow up. CONCLUSION: JCV seems to be only a bystander in the pathology of MS, and the presence of cell-free viral particles could be related to the immunological activation of the disease, mainly during relapses.

Human herpesvirus 6 and multiple sclerosis: a one-year follow-up study.

BACKGROUND: This study was undertaken in order to investigate the possible relation of HHV-6 and EBV in relapsing-remitting MS (RRMS). MATERIALS AND METHODS: A one-year follow up study was performed analysing peripheral blood mononuclear cells and serum samples of 57 patients with RRMS and 57 healthy blood donors (HBD) by a quantitative real time PCR, to detect HHV-6 and EBV. Clinical data (starting age and EDSS increase) were collected. RESULTS: We did not find any statistically significant difference for EBV between RRMS patients and HBD. Regarding HHV-6: i) There was a higher prevalence of HHV-6 in RRMS patients than in controls: 80.7% versus 29.8% respectively. ii) HHV-6 active replication seems to be related to exacerbations. iii) Only variant A was detected among RRMS patients with HHV-6 active replication. iv) Although some difference was found when we compared clinical data in RRMS patients with and without HHV-6 active replication, the results did not reach statistical significance. CONCLUSIONS: A higher HHV-6A frequency of active infection (reactivation or new infection) would lead to a more frequent exposure of HHV-6A antigens to the immune system of RRMS patients; this active replication of HHV-6A seems to be specifically related with the exacerbations in a subset of RRMS patients.