Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

Background: Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. Methods: A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data. Results: Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: p(M-H) = 0.0005; ORM-H (95% CI) = 1.27 (1.11-1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [p(M-H) = 0.0003, ORM-H (95% CI) = 1.32 (1.14-1.53)]. Conclusion: Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Human Endogenous Retrovirus (HERV) Insertional Polymorphisms

Human endogenous retroviruses (HERVs) are the result of ancient germ cell infections of human germ cells by exogenous retroviruses. HERVs belong to the long terminal repeat (LTR) group of retrotransposons that comprise ~8% of the human genome. The majority of the HERVs documented have been truncated and/or incurred lethal mutations and no longer encode functional genes; however a very small number of HERVs seem to maintain functional in making new copies by retrotranspositon as suggested by the identification of a handful of polymorphic HERV insertions in human populations. The objectives of this study were to identify novel insertion of HERVs via analysis of personal genomic data and survey the polymorphism levels of new and known HERV insertions in the human genome. Specifically, this study involves the experimental validation of polymorphic HERV insertion candidates predicted by personal genome-based computation prediction and survey the polymorphism level within the human population based on a set of 30 diverse human DNA samples. Based on computational analysis of a limited number of personal genome sequences, PCR genotyping aided in the identification of 15 dimorphic, 2 trimorphic and 5 fixed full-length HERV-K insertions not previously investigated. These results suggest that the proliferation rate of HERVKs, perhaps also other ERVs, in the human genome may be much higher than we previously appreciated and the recently inserted HERVs exhibit a high level of instability. Throughout this study we have observed the frequent presence of additional forms of genotypes for these HERV insertions, and we propose for the first time the establishment of new genotype reporting nomenclature to reflect all possible combinations of the pre-integration site, solo-LTR and full-length HERV alleles.

Protective Effect of Human Endogenous Retrovirus K dUTPase Variants on Psoriasis Susceptibility

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 x 10(-15), odds ratio = 2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis. Journal of Investigative Dermatology (2012) 132, 1833-1840; doi:10.1038/jid.2012.69; published online 22 March 2012

Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods

Abstract Background An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Results Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Conclusions Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

Genomic analysis of ERVWE2 locus in patients with multiple sclerosis: absence of genetic association but potential role of human endogenous retrovirus type W elements in molecular mimicry with myelin antigen

Human endogenous retroviruses (HERVs) arise from ancient infections of the host germline cells by exogenous retroviruses, constituting 8% of the human genome. Elevated level of envelope transcripts from HERVs-W has been detected in CSF, plasma and brain tissues from patients with Multiple Sclerosis (MS), most of them from Xq22.3, 15q21.3, and 6q21 chromosomes. However, since the locus Xq22.3 (ERVWE2) lack the 5' LTR promoter and the putative protein should be truncated due to a stop codon, we investigated the ERVWE2 genomic loci from 84 individuals, including MS patients with active HERV-W expression detected in PBMC. In addition, an automated search for promoter sequences in 20 kb nearby region of ERVWE2 reference sequence was performed. Several putative binding sites for cellular cofactors and enhancers were found, suggesting that transcription may occur via alternative promoters. However, ERVWE2 DNA sequencing of MS and healthy individuals revealed that all of them harbor a stop codon at site 39, undermining the expression of a full-length protein. Finally, since plaque formation in central nervous system (CNS) of MS patients is attributed to immunological mechanisms triggered by autoimmune attack against myelin, we also investigated the level of similarity between envelope protein and myelin oligodendrocyte glycoprotein (MOG). Comparison of the MOG to the envelope identified five retroviral regions similar to the Ig-like domain of MOG. Interestingly, one of them includes T and B cell epitopes, capable to induce T effector functions and circulating Abs in rats. In sum, although no DNA substitutions that would link ERVWE2 to the MS pathogeny was found, the similarity between the envelope protein to MOG extends the idea that ERVEW2 may be involved on the immunopathogenesis of MS, maybe facilitating the MOG recognizing by the immune system. Although awaiting experimental evidences, the data presented here may expand the scope of the endogenous retroviruses involvement on MS pathogenesis

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.

The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.

Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.

Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus

Retroviral elements are found in abundance throughout the human genome but only rarely have alterations of endogenous genes by retroviral insertions been described. Herein we report that a human endogenous retrovirus (HERV) type C is inserted in the human growth factor gene pleiotrophin (PTN) between the 5′ untranslated and the coding region. This insert in the human genome expands the region relative to the murine gene. Studies with promoter-reporter constructs show that the HERV insert in the human PTN gene generates an additional promoter with trophoblast-specific activity. Due to this promoter function, fusion transcripts between HERV and the open reading frame of PTN (HERV-PTN) were detected in all normal human trophoblast cell cultures as early as 9 weeks after gestation (n = 7) and in all term placenta tissues (n = 5) but not in other normal adult tissues. Furthermore, only trophoblast-derived choriocarcinoma cell lines expressed HERV-PTN mRNA whereas tumor cell lines derived from the embryoblast (teratocarcinoma) or from other lineages failed to do so. We investigated the significance of HERV-PTN mRNA in a choriocarcinoma model by targeting this transcript with ribozymes and found that the depletion of HERV-PTN mRNA prevents human choriocarcinoma growth, invasion, and angiogenesis in mice. This suggests that the tissue-specific expression of PTN due to the HERV insertion in the human genome supports the highly aggressive growth of human choriocarcinoma and possibly of the human trophoblast.

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats

Background: Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (βERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses.Results: A diverse range of full-length βERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years.Conclusions: Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of βERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health. © 2013 Hayward et al.; licensee BioMed Central Ltd.

Human endogenous RNAs: Implications for the immunomodulation of Toll-like receptor 3

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFN gamma and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3(+), CD4(+) and CD8(+) cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBMCs treated with endogenous RNA was down-regulated 1.8-fold (p=0.002). The same trend was observed for IFN gamma where in the presence of poly(I:C) an 8.7-fold increase was noted and in the presence of endogenous RNA a 3.1-fold decrease was observed. In the culture activated with poly(1:C), mRNA expression of CXCR4 increased 8.0-fold and expression of PKR increased 33.0-fold. Expression of these genes decreased in the culture treated with endogenous RNA when compared to the culture without stimulus. Thus, high expression of mRNA for TLR3, IFN gamma, CXCR4 and PKR was observed in the presence of poly(I:C) and low expression was observed in the cells cultured with endogenous RNA. In conclusion, TLR3 may play major physiological roles that are not in the context of viral infection. It is possible that RNA released from cells could contain enough double-stranded structures to regulate cell activation. The involvement of endogenous RNA in endogenous gene expression and its implications in the regulation thereof, are still being studied, and will have significant implications in the future.