ERV3 Env Research Articles

Abstract A55: Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.

Abstract Purpose: The first aim is to investigate the activation of 7 immune checkpoints at different time points following transfection of three diverse ERV envelope (env) genes in serous ovarian carcinoma cells. The second aim is to determine if the ERV-W1 Syncytin-1 env and ERV-3 protein are expressed in primary serous OvCa tumors and ascites fluids to explore a possible role in the tumor microenvironment. Introduction: The overall survival of serous OvCa has not greatly improved. We and others have demonstrated up-regulation of interferon (IFN) type I signaling pathway viral defense genes including IFN stimulated genes (ISGs), in a variety of human tumors (ovary, breast, lung) but also in ovarian and colon carcinoma cell lines treated with the DNA methyltransferase inhibitor Aza-C. Aza-C treatment of serous OvCa cell lines leads to an induction of PD-L1 expression as mediated downstream signaling via IFNg. We recently demonstrated that activated ERVs function at the RNA level to ignite the IFN response. Exogenous retroviruses like HIV have evolved to suppress the immune response by up-regulation of PD-L1 on target cells. Tumors mimic regulatory mechanisms involving immunosuppression and inflammation, which we propose involves ERVs. Experimental Procedures: ERV-3, Syncytin-1 and ERV-W2 env CMV overexpression vectors and a control EGFP-vector were transfected into OvCa serous cell lines (TykNu, A2780 and Hey) and RNA expression of 7 immune checkpoint molecules (LGALS9, PDL-2, CD80, CD276, PD-L1, VTCN1 and PD1) were monitored at days 3 and 7 post transfection using real time PCR. Immunohistochemistry (IHC) and Western analyses using antibodies for Syncytin-1 and ERV-3 were performed with primary serous OvCa tumors. Western and ELISA analyses of serous OvCa ascites were executed to detect Syncytin-1 protein levels. Syncytin-1 protein concentrations were quantified in ascites with two different antibodies using ELISA and correlated to a standard curve based on purified Syncytin-1 levels. Results: Three serous OvCa cell lines following ERV env transfection showed differences of immune checkpoint activation. TykNu demonstrated the highest fold-levels of induction for LGALS9 (73.1x), PD-L1 (15.2x), PD-L2 (9.8x) and PD1 (6.1x) at day 3 for all ERV expression vectors, which decreased by day 7 except for PD1. A2780 showed increased levels for LGALS9 (7.2x) and PD1 (5.2x) with all ERV expression vectors at day 3, which decreased by day 7. In contrast, Hey cells showed no significant increase of the analyzed immune checkpoint genes. Syncytin-1 and ERV-3 protein was highly expressed in primary OvCa epithelial cells using IHC, compared to patient matched control ovarian tissues. Western analyses of ascites from OvCa patients showed high levels of Syncytin-1 in the supernatant, supporting protein secretion. Absolute quantification using real time PCR of Syncytin-1 and ERV-3 RNA with fractionated OvCa ascites cells demonstrated high amounts of molecules. ELISA results confirmed high levels of Syncytin-1 (98.6 - 525.8 microg/ml) in ascites of primary OvCa and in one relapse patient (413.4 microg/ml), but were virtually undetectable in an ascites from a gastro-intestinal carcinoma and control peritoneal fluids (2.7 microg/ml). Conclusions: Our findings that various immune checkpoints are differentially regulated following ERV activation in serous OvCa cell lines endorses that analyses of immune checkpoints prior to treatment could help stratify patient therapy. ERVs like exogenous retroviruses not only play an essential functional role at the RNA level via TLR3, but also possibly at the protein level via TLR4 modulation of immune / inflammatory responses. Citation Format: Pamela L. Strissel, Katherine B. Chiappinelli, Johanna Strehl, Franziska MB Würfel, Matthias W. Beckmann, Stephen B. Baylin, Reiner Strick. Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A55.

Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies.

Fusion, proliferation, angiogenesis, immune tolerance, and tissue survival are some of the critical functions involved in the physiological and pathological processes of placenta development. Strikingly, some of these properties are shared by envelope glycoproteins of retroviruses. Part of the overall retroviral world, the human retroviral heritage consists of hundred thousands of elements representing a huge amount of genetic material as compared to our 25,000 genes, whereas only a few tenths of retroviral loci still contain envelope genes exhibiting large open reading frames. Some of these envelopes, namely Syncytin-1, Syncytin-2, and ERV-3 Env, were shown to support essential functions in placenta development. First, in order to understand where these envelope genes originate and what are the critical mechanisms involved in transcription regulation and protein basic functions such as recognition of cellular receptor by viral envelopes, we will describe the retroviral life cycle and how repeated infections during species evolution led to the formation of retroviral families. We will emphasize how many envelope genes remain in our genome and in which organs they were found to be expressed. Second, Syncytin-1 will be used as a model to decipher essentially in placental context (i) the detailed modalities of transcriptional control including repressive histone marks and CpG methylation epigenetic mechanisms, involvement of tissue-specific transcription factors, and control of mRNA splicing, as well as (ii) the multiple steps required for protein maturation finally leading to a functional trimeric glycosylated protein. The extraordinary versatility of Syncytin-1 will permit to demonstrate that such proteins are likely involved in physiological processes not only in placenta but also in other organs, based on evidence of fusion/differentiation, immunomodulation, apoptosis, and proliferation properties. Third, we will describe extensively the altered behavior of the various levels of transcriptional control or of protein functions/localization/maturation displayed by Syncytins and other endogenous retroviral envelopes. We will exemplify how such altered states may contribute to human placenta pathologies, including Down syndrome, preeclampsia/hemolysis, elevated liver enzymes, and low platelets syndrome/intrauterine growth restriction, and gestational trophoblastic diseases including mole and choriocarcinoma. Similar deregulations will be respectively mentioned on this target of fetal invasion that is the endometrium, the reproductive organs that are the testis and the ovary, and in the breast nourisher of the newborn child. All these observations draw outlines of the symbiotic and conflicting mechanisms at work where the retrovirus world and the human world have converged.

Transfection of BeWo cells with a truncated human endogenous retrovirus ERV3 env induces β-hCG

Transfection of BeWo cells with a truncated human endogenous retrovirus ERV3 env induces β-hCG

Evidence for expression of endogenous retroviral sequences on primate reproductive tissues and detection of cross-reactive ERVS antigens in the baboon ovary: a review

To review recent research findings on the specific expression of endogenous retroviral sequences (ERVS) in reproductive tissues and their possible physiological roles. ERVS have been implicated in several biological events such as induction of resistance to exogenous retrovirus invasion, involvement in placental trophoblast formation, sperm maturation and differentiation; and stimulation of local immunosuppression to protect the foetus from immunological attack. Critical review of relevant articles and abstracts cited in international and local journals, literature searches on Medline and Medchem up to 2005. Retroviruses have been implicated in the induction of tumour and immunological disorders. Over the years, endogenous retroviruses (ERVs) and retroviral elements have been detected in the genome of many vertebrate species, including primates. The evidence for the presence of retroviruses in the primate tissues such as the placenta, ovary, breast, testis and epididymis has been documented using electron microscopic studies. Retrovirus-like particles were found budding from the basal membrane of syncytiotrophoblasts, as well as in tumour cell lines in embryonic carcinoma or teratocarcinomas. Apart from their pathological effects, recent evidence suggests that these ERVs may play useful roles in normal physiological events. Recent studies indicate the expression of endogenous retroviruses in the testis, epididymis, placenta and breast. However, limited data exist on the detection of ERVs in the ovary. Overall, the precise functions for ERVs in these tissues are not well understood. In the testis and epididymis, speculative functions may include among others spermatogenesis and/or sperm maturation (differentiation) whereas in placenta they are possibly associated with trophoblast fusion and locally induced immunosuppression to protect the foetus from immunological attack. Experiments in our laboratory have indicated restricted expression of retroviral antigens including baboon endogenous retroviral proteins (BERV), ERV-3, HIV-1 gp41 and HERV-K env in the baboon ovary. ERVs are specifically expressed in different mammalian reproductive tissues and may have unique physiological roles.

Developmental Expression of HERV-R (ERV3) and HERV-K in Human Tissue

The human endogenous retroviruses (HERVs), ERV3 (HERV-R) and HERV-K, are both known to be transcriptionally active in human placenta. In the case of ERV3 there is also indirect evidence for its participation in cellular differentiation. In this study we examined the expression of ERV3 (HERV-R) and HERV-K in human normal fetal tissues by in situ hybridization. The highest level of ERV3 env expression was detected in primitive adrenal cortex. Elevated levels of expression were also found in the following developing tissues: kidneys (tubules), tongue, heart, liver, and central nervous system. Tissue-specific expression was found for HERV-K rec (former cORF) but not for pol/int transcripts. The highest rec expression was found in placenta and levels slightly higher than sense control were found in the rest of the tissues examined. Pol/Int was not possible to quantitate. It appears that ERV3 is expressed in an organ-specific way during embryogenesis and might suggest a possible role in the development and differentiation of human tissues.

Endogenous retrovirus sequences expressed in male mammalian reproductive tissues: a review.

To review the research findings on the expression of endogenous retroviruses and retroviral-related particles in male mammalian reproductive tissues, and to discuss their possible role in normal cellular events and association with disease conditions in male reproductive tissues. Published findings on endogenous retrovirus (ERV) expression in vertebrate reproductive tissues. Relevant citations on ERVs and male reproduction by research groups worldwide. Literature search on Medline and Pubmed upto the year 2000, and retrieval of relevant articles cited from international and local journals. Most of the studies demonstrated integrated retroviruses and retroviral-related sequences in human and mouse testis, epididymis and vas deferens. Endogenous retroviruses in human and mice may be associated with normal cellular differentiation and development, and carcinogenesis. In humans, one ERV family, human endogenous retrovirus-K (HERV-K) is abundantly expressed, and is associated with germ cell tumours, while ERV3 env is expressed in normal human testis. The expression of ERVs in male reproductive tissues suggests a possible role in normal and disease conditions involving the testis and epididymis. These speculative functions may include among others spermatogenesis and or sperm maturation or tumour formation. However, further studies need to be carried out to investigate specific roles of ERVs in male reproductive events.

The Cellular Mechanism by which the Human Endogenous Retrovirus ERV-3 env Gene Affects Proliferation and Differentiation in a Human Placental Trophoblast Model, BeWo

The env region of the human endogenous retrovirus ERV-3 is expressed during differentiation of trophoblast and the choriocarcinoma BeWo. Stable transfectants with ERV-3 env exhibit most aspects of trophoblast differentiation, including inhibition of cell proliferation, changes in cell morphology, and increased production of β-hCG mRNA. In this study, the cellular mechanism of induction of BeWo cell differentiation by ERV-3 env was investigated. In BeWo cells stably transfected with ERV-3 env, the production of β-hCG mRNA and hCG protein was increased. Intracellular cAMP level was markedly increased over that of vector transfected cells. The effect on β-hCG protein production was inhibited by H89, a protein kinase A (PKA) inhibitor, while protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors had no effect. The expression of a major cell cycle promoter, cyclin B, was markedly reduced while expression of p21, a negative regulator of the cell cycle, was up-regulated. Inhibition of ERV-3 env induced hCG production with H89 had no significant effect on cell growth when compared with cells transfected with vector alone.

The expression of ERV-3 mRNA in a choriocarcinoma model of trophoblastic differentiation

ERV-3 is a human endogenous retrovirus genome that has been isolated and sequenced. The expression of ERV-3 mRNA and its protein in the syncytiotrophoblast of the placenta suggests that ERV-3 proteins may play a role in placental development. The choriocarcinoma cell line BeWo, a model of trophoblast differentiation, is maintained in cell culture in an undifferentiated and non-fused state. Upon addition of forskolin, a cAMP inducing agent, BeWo cells undergo intercellular fusion and differentiation. In untreated BeWo, low level expression of ERV-3 env -specific mRNA was only detected by RT-PCR. Northern blot analysis showed that the expression of 3.5 kb ERV-3 env -specific gene was increased significantly in BeWo cells after 48 hours forskolin treatment and decreased at 96 hours. The up-regulation of ERV-3 env -specific gene was concurrent with the production of human chorionic gonadotropin (β-hCG) mRNA, a trophoblast differentiation marker. In situ hybridization studies revealed that the peak expression of ERV-3 env mRNA occurred at 72 hours after forskolin treatment, when about 80% of cells were fused to form multinuclear syncytia. These results indicate that the expression of ERV-3 env -specific mRNA is up-regulated during forskolin-induced BeWo cell differentiation, suggesting a role for ERV-3 gene products in that process.

Expression of the endogenous retrovirus ERV3 (HERV-R) during induced monocytic differentiation in the U-937 cell line

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. LTR-env-gene-spliced mRNA of 9 and 3.5 Kb is widely expressed in human tissues and cells, but gag-pol mRNA has not been found. Further, the env gp70 gene contains an open reading frame throughout its length and its expression has recently been detected as a full-length protein. The highest expression of ERV3 detected so far is in placenta and the lowest in cytotrophoblasts and choriocarcinoma cell lines. In this report we have studied ERV3 mRNA and protein expression in the human monoblastic cell line U-937 during differentiation into monocytes/macrophages. Differentiation of U-937 cells was induced by 1,25a-dihydroxyvitamin D3 (vitD3), retinoic acid (RA), gamma interferon (IFN-gamma) and phorbol-myristate-acetate (PMA-TPA). The expression of ERV3 env mRNA was found to be differentiation-associated, with high expression detected in the late stages of monocytic development. Using TPA, the expression of ERV3 env was detected as 9- and 3.5-kb transcripts by Northern blotting, as mRNA by in situ hybridization and as a cytoplasmic 65-kDa protein by immunofluorescence and Western blots. Low levels of basal expression were found, with up-regulation of both message and protein at 24 to 48 hr after addition of TPA. Induction with vitD3, IFN-gamma and RA produced higher levels of mRNA at earlier time points. It is concluded that the U-937 cell line represents an excellent model system for further studies to study the relationship between ERV3 expression and cellular differentiation.

Elevated Levels of the Endogenous Retrovirus ERV3 in Human Sebaceous Glands

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. Long terminal repeat-envelope (env) gene spliced mRNAs of 9 and 3.5 kb are widely expressed in human tissues and cells, but gag-pol mRNAs have not been found. Furthermore, the env gp70 gene contains an open reading frame throughout its length. The highest expression of ERV3 mRNA detected so far is in placenta and the lowest in choriocarcinoma cell lines. We have previously shown that the human monoblastic cell line U-937 and some normal and neoplastic tissues also express high levels of ERV3 env message by Northern blot analysis; however, this method does not distinguish between mRNA expression in different cell types in tissues. In this report, we have studied the ERV3 mRNA expression in specific cell types of human skin by in situ hybridization. We found high levels expression of ERV3 env mRNA in human sebaceous glands in normal skin and dermoid cysts of the ovary. In all glands, the expression is maximal in the periphery of the lobule and ceases towards the center in the region of characteristic holocrine secretion. Since it is known that the regulation of sebaceous glands is primarily via steroid hormones, particularly androgens, it is possible that expression of ERV3 is hormone dependent.

Abundance of an Endogenous Retroviral Envelope Protein in Placental Trophoblasts Suggests a Biological Function

To investigate the hypothesis that the human endogenous sequence ERV-3 has a function, we have cloned and expressed the transmembrane region of its envelope gene and raised specific antibodies to the fusion protein and to a synthetic peptide. These antibodies reacted with a 65-kDa polypeptide which constituted approximately 0.1% of the cellular protein in syncytiotrophoblasts in placenta. The evolutionary conservation and abundant expression of this endogenous retroviral protein in a specific cell type support the concept of a biological function. The similarity of a domain of ERV-3 env to putative immunosuppressive p15E sequences suggests that ERV-3 might form part of the placental immunosuppressive barrier between mother and foetus.

Expression of an endogenous retrovirus (ERV3 HERV-R) in human reproductive and embryonic tissues--evidence for a function for envelope gene products.

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. It is expressed in several human tissues as LTR env spliced transcripts (9 and 3.5 kb). The highest level of expression is to be found in placenta and virus expression is down-regulated in choriocarcinoma cell lines. By means of in situ hybridization, the expression of ERV3 env was studied in selected human reproductive and embryonic tissues. It is concluded that (a) ERV3 env is expressed in syncytiotrophoblasts not only in the placenta but also in hydatidiform moles and choriocarcinomas (irrespective of origin) (b) ERV3 expression in placenta correlates to cell fusion but probably not to the fertilization process itself (c) ERV3 env is highly expressed in certain cells in spermatogenesis but not in the Sertoli or Leydig cells, and finally (d) ERV3 env is expressed in certain embryonic tissues such as the adrenal gland and nervous tissues.