The Role of Endogenous Retroviruses in Placental Gene Expression: Difference between revisions

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==Function of <i>env</i> ERV- 3 Protein==
==Function of <i>env</i> ERV- 3 Protein==
[[Image: ENVglycoprotein.jpeg|thumb|220px|left|Figure 7:</b> Functional Domains of Env Glycoproteins Proposed to be Involved in Placental Morphogenesis. Ev-3 codes for a truncated Env in humans. Synctin-2 (human) and Synctin-B (murine) has all possible components of a functional Env, including: leader peptide, furin cleavage site, fusion peptide, ISD, membrane spanning domain and a cytoplasmic tail.<ref name = Black2010/>]]
[[Image: ENVglycoprotein.jpeg|thumb|220px|left|Figure 7:</b> Functional Domains of Env Glycoproteins Proposed to be Involved in Placental Morphogenesis. Ev-3 codes for a truncated Env in humans. Syncytin-2 (human) and Synctin-B (murine) has all possible components of a functional Env, including: leader peptide, furin cleavage site, fusion peptide, ISD, membrane spanning domain and a cytoplasmic tail.<ref name = Black2010/>]]


In addition to  HERV-W and and HERV-FRD, a third Endogenous Retrovirus, Erv-3, is an important gene related to the role of ERVs present in the placenta. With respect to the structure, Env-3 has a long open reading frame, it is unique in that it has a premature stop codon which inhibits its ability to anchor to the cell membrane. Furthermore, Erv-3 lacks a leader and fusion peptide (<b>Figure 7</b>).<ref name=Black2010/> Researchers have used BeWo cells (from a choriocarcinoma) to model trophoblast differentiation. In one study, the expression of Erv-3 mRNA was observed over a span of 96 hours with treatment of forskolin in order to identify how this gene functions in comparison to the prominent genes (HERV-W and HERV-FRD) which code for Syncytin proteins. In a separate test,  researchers constructed expression vectors containing either the sense strand of the Erv-3 env open reading frame or only the plasmid.  
In addition to  HERV-W and and HERV-FRD, a third Endogenous Retrovirus, Erv-3, is an important gene related to the role of ERVs present in the placenta. With respect to the structure, Env-3 has a long open reading frame, it is unique in that it has a premature stop codon which inhibits its ability to anchor to the cell membrane. Furthermore, Erv-3 lacks a leader and fusion peptide (<b>Figure 7</b>).<ref name=Black2010/> Researchers have used BeWo cells (from a choriocarcinoma) to model trophoblast differentiation. In one study, the expression of Erv-3 mRNA was observed over a span of 96 hours with treatment of forskolin in order to identify how this gene functions in comparison to the prominent genes (HERV-W and HERV-FRD) which code for Syncytin proteins. In a separate test,  researchers constructed expression vectors containing either the sense strand of the Erv-3 env open reading frame or only the plasmid.  

Revision as of 02:15, 8 April 2021

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By Anna Harnsberger

Introduction

The role of integrated or “endogenous” retroviruses (ERVs) is extensive. ERVs contribute both positively and negatively toward the health of humans and other taxonomic groups. The functionality of these viruses relies heavily on the process of retrotransposition. This process involves the reverse-transcription of the retrovirus genome into the DNA of the host cell. Though some ERVs are detrimental to human health, such as HIV-1, others are “domesticated” and do not pose a threat.[1] Domesticated ERVs, likely inactivated by mutations over millions of years, serve in adaptive functions of the host. [2] Specifically, the presence of ERVs are essential during human pregnancy. The placenta, a vital organ developed during gestation, is affected by the presence of ERVs. ERVs, acting as non-coding regulatory elements, are key contributors to placental evolution. Absence or mis-expression of specific ERVs in the placenta during pregnancy, results in pre- and or post- defects such as pre-eclampsia (PE). Despite what is known about the importance of ERVs in placental gene expression, there is still much research to be done to fully understand the impacts of these retroviruses on the placenta.

The Role of Endogenous Viruses in the Placenta

ERVs are an essential component of the development of the placenta, not only for humans but a vast array of animals. The origin of ERVs into the placenta is understood to have occured approximately 100 million years ago (Myr). ERVs parasitized living species even before the viruses began to appear in mammals.[3] As opposed to exogenous viruses which are associated with infection, ERVs are germ-line integrated into the host DNA by vertical gene transfer.[4] ERVs evolve more slowly than their exogenous counterparts and function in a sort of symbiosis with their host, given their normal presence in the genome. The importance of ERVs in the placenta focuses on specific envelope genes that originate from the retrovirus, these will be covered in depth in the following sections. The core components to consider when understanding the importance of ERVs is their role in cell differentiation, cell fusion and their immunosuppressive properties.

Placental & Endogenous Retrovirus Structural Components

Figure 1: (A) The General Structure of an ERV with (B) Core Encoded Proteins, and (C) a lone Long Terminal Repeat (LTR) (characteristic of env genes).[5]

The presence of ERVs in host genomes is the result of either direct insertion by host homologous end joining, or from transposition through the use of retrotransposons.[3] Approximately 8% of the human genome consists of endogenous retroviral elements.[6] Although exogenous retroviruses and ERVs have different impacts on the human body, they share the same structure which consists of four viral genes: gag, pro, pol and env, flanked by two long terminal repeats (Figure 1). Gag genes encode for major viral structural proteins; pro and pol encode for viral enzymatic machinery and Env encodes the envelope glycoprotein. The envelope glycoprotein embeds into the lipid bilayer of the exterior membrane in order to form the viral envelope and controls the entry of viruses into susceptible cells.[7] These components of the envelope protein are significant in the way which ERV evolution occurred in mammals and the way in which envelope genes can affect the different functional components of mechanisms within the placenta. The placenta itself has several key structural components which are important to understand.

Nomenclature to understand with respect to this topic includes the placental tissue, also known as the trophoblast, composed of peripheral cells of the blastocyst. The blastocyst is a specific stage of the mammalian embryo, with a composition similar to that of a hollow cell ball. Furthermore, cytotrophoblasts are important for attachment to the inner uterine wall. These cells are important for cell-cell fusion along with myoblasts, macrophages, and gametes. Most notably, cytotrophoblasts are important in formation of syncytiotrophoblasts. Syncytiotrophoblasts aid in mediating implantation of the embryo into the maternal endometrium as well as functions of exchange related to the mother and fetus, regulatory immune response, protection against pathogens and hormone secretions.[3] The terminology related to placental development is necessary to understand, with respect to how ERVs affect the organ.

The Surface and Transmembrane Subunits

Figure 2: The Retroviral Envelope Glycoprotein. (a) Structure of retroviral envelope protein with TM and SU subunit. (b) Interaction between a retroviral envelope protein and its cognate receptor: i) Virion-cell membrane fusion and virus entry into a specific cell and ii) Cell-cell membrane fusion and formation of a synctium.[3]

The different components of the retroviral envelope consists of the surface (SU) and transmembrane (TM) subunits as well as the immunosuppressive domain (ISD).[3] The env gene of retroviruses encodes for a protein which is cleaved by a cellular protease in order to produce the TM and SU subunits. The SU subunit has a domain which interacts with a receptor on the surface of cells that is only susceptible with the virus. The TM subunit is responsible for triggering the fusion of the virus and cell membranes by insertion of its N-terminal fusion peptide into the cell membrane.[3] These subunits remain as a heterodimer at the surface of the virion envelope membrane (Figure 2). The role of these subunits is important with respect to the immunosuppressive activity of the Syncytin-2 protein.

The Evolutionary Diversification of the Placenta

Figure 3: Endovascular Trophoblast Invasion in Human Pregnancy Before Week 6 of Invasion. (A) Post week 20, normal invasion. (B) Post week 20, after PE and or IUGR. (C) Failure of endovascular trophoblast invasion in PE and IUGR is specific to the placenta bed and does not affect segments of uteroplacental arteries. Blue circles represent fetal tissue; Red lines represent maternal tissue; ps= placental separation. Ps indicates separation between basal plate (attached to placenta) and placental bed (remains in placenta after delivery). [8]

Despite the emergence of ERVs in the placenta after the evolution of mammals, there are multiple factors which lead to differentiation of the placenta amongst these different species. Including diversity of the cytotrophoblasts, syncytiotrophoblasts and the degree of invasiveness of trophoblasts. Amongst different species, there is variation of the types of placental interface. There is an increase of invasive trophoblasts from synepitheliochorial (ruminants), endothelialcorial (carnivores), and hemochorial (mice, rabbits, humans). Trophoblast invasion is important in all of these species, since malinvasion can result in PE or Intrauterine growth retardation (IUGR). (Figure 3)[8]

Synctin: Key Proteins Originating from Endogenous Retroviruses

When considering the role of ERVs on the development and regulation of the placenta, the key proteins that should be considered are “Synctins. Synctins are cellular proteins which correspond to functional ERV env open reading frames as single copies, which have been conserved throughout evolution.[9] Syncytin proteins play an important role in cell-cell fusogenic activity and immunosuppression of the envelope proteins. [3]

Figure 4: (a) Phylogenetic Tree of Mammals with Four Major Groups of Eutherians. Four groups of eutherians include: I) Afrotheria, II) Xenartha, III) Euarchontoglires and IV) Laurasiatheria. The timeline indicates the insertion time of the syncytin genes. (b) Represents types of placental diversity, color coded outline of the diagrams correspond with a given type of placentation depicted in (a). Placental types organized from top to bottom in order of decreasing invasion properties and syncytialization[3]

There are two notable types of Syncytin proteins found in the human placenta, encoded by Env genes HERV-W (Syncytin-1) and HERV-FRD (Syncytin-2). Syncytin-1 is a glycoprotein, with cell fusogenic activity, promoting the fusion of cytotrophoblast cells in order to form the multinucleated syncytiotrophoblast layer. Syncytin-2 is associated in its role of immunosuppression.[5] The membrane receptor for Syncytin-1 is ASCT-2 and ASCT-1 and the phospholipid transporter MFSD-2 for Syncytin-2.[9] The functionality of these two genes has been conserved in evolution since their first insertion into the primate genome, approximately 25-40 Myr, Syncytin-2 is the more ancient of the two genes. Both genes have little polymorphism and therefore show signs of purifying selection or stabilizing selection. [3] Analogous to the human “Syncytin-1” is the murine gene “Synctin-A”, the human “Synctin-2” gene is analogous to the murine “Synctin-B” gene. It is important to note that although the murine and human Syncytin proteins are similar in their functions, they are homologous but not orthologous(Figure 4).[3]

Fusogenicity of Syncytin-A and Syncytin-1

Figure 5: Both Murine Syncytin Genes are Needed for Syncytiotrophoblast Double Layer Formation. (a) Representation of fetal maternal interface of ST-I and ST-II. (b) Electron Microscopy image of placental feto-maternal interface of mice lacking Syncytin-A or Syncytin-B (ii).[3]

Given the likeness of the murine Syncytin proteins with those of humans, murine have been a beneficial model for determining the role that Syncytin proteins play in placental development. It has been found that using the mouse model, that synctin-B and Gcm1 (the placental transcription factor) genes are both expressed in the ST-II layer, one of the two distinct layers of syncytiotrophoblasts within mice. [3] The two ST-layers arise from the fusion of trophoblast cells during the “labyrinth formation”(Figure 5).[3] The labyrinth is the interface for nutrient and other gas exchange between the embryo and mother.[10] The ST-II layer contains an abundance of lipids which are tightly adhered to one another through gap junctions available for intracellular transport.[11]

Syncytin-A was found to be expressed in the other layer, ST-I. Using knock-out gene expression, it was found that loss of Syncytin-A gene in mice was detrimental, homozygous mutant embryos died mid-gestation. This demonstrates that lack of Syncytin-A mediated trophoblast cell-fusion results in cell overexpansion affecting the fetal blood vessel spaces due to apoptosis, and leading to defects of the maternal-fetal interface structure causing decreased vascularization and inhibition of placental transport.[11] Absence of Syncytin-A ultimately results in the death of the embryo. Thus, it can be conferred that much like Syncytin-A, Syncytin-1 is vital to embryo survival (Figure 6). This study highlights that a gene of retroviral origin is necessary for found physiological processes of the placenta.[10]

Figure 6: Morphological and Physiological Abnormalities of SynA Null Embryos. (A) Mean fetal weights of wild-type, heterozygous and SynA null embryos at varying embryonic stages. (B)-(E) Vascularization of E13.5 wild type(left) and SynA null (right) embryos. (B) SynA null embryo is smaller and more pale in comparison to the wild type. (C) Yolk sac surrounding the embryo, arrows point to blood vessels. Blood vessels of the SynA Yolk Sac are barely visible, the yolk sac surrounding wild type embryos has distinct blood vessels.(E) Fetal side of placenta, less vascular for mutant embryo.(F) Transplacental passage of Rhodamine 123 into E11.5 embryos, post 2 hr i.p injection of the fluorescent dye into the mother. [11]

Furthermore, researchers found that both homotypic and heterotypic fusion was promoted via the expression of HERV-W in placenta specific cells. With respect to homotypic fusion, cell-cell fusion assays which were transiently expressed in parental TE671 human cells resulted in an abundance of syncytia.[12] Heterotypic fusion was demonstrated by cocultivating TE671 Env-transfected cells with HeLa indicator cells. Post 48 hours, up to 48 % of the coculture had distributed into large syncytia. In comparison, expression of A-Rless (a mouse leukemia virus) resulted in fusion of only ~6% of the cultured cells (Fig 8) (Blond et al., 2000). These findings support that Syncytin-1 protein, which results from expression of HERV-W, is a highly fusogenic membrane protein. However, it is important to note that the fusogenic property of this HERV-W expressed protein depends on the interaction with specific cell surface receptors.[12]

Immunosuppression of Syncytin-B and Syncytin-2

In contrast to the role of the Syncytin-1 protein of cell-cell fusion , Syncytin-2 has immunosuppressive properties that are encoded by retroviral envelope sequences. Though Syncytin-2 aids in the fusion of the placental trophoblasts as well as the formation of multinucleated syncytiotrophoblast, it has been found to play a more prominent role in immunosuppression..[13] Researchers, have focused on the ISD of the Syncytin-B protein using deletion analysis of immunosuppressive and non immunosuppressive sequences to discover the mutation of amino acids which causes the switch from immunosuppressive to the non immunosuppressive envelope protein.[14] Researchers used in vivo tumor rejection-assays to demonstrate the IS activity of env proteins in which expression of the immunosuppressive Syncytin protein enabled the temporary engraftment of allogeneic tumor cells. Furthermore, IS activity of Syncytin-2 was further examined an another in vivo assay that used recombinant Env subdomains, primarily ectodomains (domain of a membrane protein extending outside the cell) to demonstrate enhanced antibody response against the Env protein after inhibition of the ISD function.[14] IS activity of the Syncytin-2 is essential for development of the fetus, its IS activity is likely most necessary for protecting the developing fetus from the surveillance of the maternal immune system.[15]

Function of env ERV- 3 Protein

Figure 7: Functional Domains of Env Glycoproteins Proposed to be Involved in Placental Morphogenesis. Ev-3 codes for a truncated Env in humans. Syncytin-2 (human) and Synctin-B (murine) has all possible components of a functional Env, including: leader peptide, furin cleavage site, fusion peptide, ISD, membrane spanning domain and a cytoplasmic tail.[7]

In addition to HERV-W and and HERV-FRD, a third Endogenous Retrovirus, Erv-3, is an important gene related to the role of ERVs present in the placenta. With respect to the structure, Env-3 has a long open reading frame, it is unique in that it has a premature stop codon which inhibits its ability to anchor to the cell membrane. Furthermore, Erv-3 lacks a leader and fusion peptide (Figure 7).[7] Researchers have used BeWo cells (from a choriocarcinoma) to model trophoblast differentiation. In one study, the expression of Erv-3 mRNA was observed over a span of 96 hours with treatment of forskolin in order to identify how this gene functions in comparison to the prominent genes (HERV-W and HERV-FRD) which code for Syncytin proteins. In a separate test, researchers constructed expression vectors containing either the sense strand of the Erv-3 env open reading frame or only the plasmid.

It was found that BeWo cells that contained the vector, had significant depression of cell growth (detected using 3H-tdR). BeWo transfected with just the plasmid had no evident morphological changes.[16]

In both the study using the forskolin induced cells and the vector containing the Erv-3 env open reading frame, there was increased production of β-hCG (a hormonal differential marker for trophoblasts). Furthermore, Erv-3 env was expressed in undifferentiated BeWo cells, inducing changes similar to those of trophoblast differentiation. Thus it was thought that env protein Env-3 had a greater role in cell differentiation rather than cell-cell fusion.[16] However, 1 % of caucasian individuals have the previously mentioned premature stop codon in the Erv-3 gene, rendering the protein non-functional. [7] Thus, the env protein Env-3 serves as a good example in the way which ERVs have evolved to be mutationally disabled within the normal tissues of humans, specifically in reproductive organs.





Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2021, Kenyon College.

  1. ↑ [Haig, D. (2012). Retroviruses and the placenta. Current Biology,22(15), R609-R613.]
  2. ↑ [Knox, K., & Baker, J. C. (2008). Genomic evolution of the placenta using co-option and duplication and divergence. Genome research, 18(5), 695-705. ]
  3. ↑ 3.00 3.01 3.02 3.03 3.04 3.05 3.06 3.07 3.08 3.09 3.10 3.11 3.12 [Lavialle, C., Cornelis, G., Dupressoir, A., Esnault, C., Heidmann, O., Vernochet, C., & Heidmann, T. (2013). Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation. Philosophical Transactions of the Royal Society B: Biological Sciences, 368(1626), 20120507. ]
  4. ↑ [Harris J. R. (1998). Placental endogenous retrovirus (ERV): structural, functional, and evolutionary significance. BioEssays : news and reviews in molecular, cellular and developmental biology, 20(4), 307–316.]
  5. ↑ 5.0 5.1 [Soygur, B., & Sati, L. (2016). The role of Syncytin proteins in human reproduction and reproductive organ cancers, Reproduction, 152(5), R167-R178. Retrieved Apr 4, 2021, from https://rep.bioscientifica.com/view/journals/rep/152/5/R167.xml]
  6. ↑ [Griffiths, D. J. (2001). Endogenous retroviruses in the human genome sequence. Genome biology, 2(6), 1-5. ]
  7. ↑ 7.0 7.1 7.2 7.3 [Black, S. G., Arnaud, F., Palmarini, M., & Spencer, T. E. (2010). Endogenous retroviruses in trophoblast differentiation and placental development. American journal of reproductive immunology (New York, N.Y. : 1989), 64(4), 255–264. https://doi.org/10.1111/j.1600-0897.2010.00860.x ]
  8. ↑ 8.0 8.1 [Kaufmann, P., Black, S., & Huppertz, B. (2003). Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biology of reproduction, 69(1), 1-7. ]
  9. ↑ 9.0 9.1 [Coquin, Y., Ferrand, M., Seye, A., Menu, L., & Galy, A. (2019). Syncytins enable novel possibilities to transduce human or mouse primary B cells and to achieve well-tolerated in vivo gene transfer. bioRxiv, 816223. ]
  10. ↑ 10.0 10.1 [Tai-Nagara, I., Yoshikawa, Y., Numata, N., Ando, T., Okabe, K., Sugiura, Y., Ieda, M., Takakura, N., Nakagawa, O., Zhou, B., Okabayashi, K., Suematsu, M., Kitagawa, Y., Bastmeyer, M., Sato, K., Klein, R., Navankasattusas, S., Li, D. Y., Yamagishi, S., & Kubota, Y. (2017). Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling. Development (Cambridge, England), 144(13), 2392–2401.https://doi.org/10.1242/dev.149757]
  11. ↑ 11.0 11.1 11.2 [Dupressoir, A., Vernochet, C., Bawa, O., Harper, F., Pierron, G., Opolon, P., & Heidmann, T. (2009). Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proceedings of the National Academy of Sciences, 106(29), 12127-12132. ]
  12. ↑ 12.0 12.1 [Blond, J. L., Lavillette, D., Cheynet, V., Bouton, O., Oriol, G., Chapel-Fernandes, S., ... & Cosset, F. L. (2000). An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. Journal of virology, 74(7), 3321-3329.]
  13. ↑ [Toufaily, C., Lokossou, A. G., Vargas, A., Rassart, É., & Barbeau, B. (2015). A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model. PloS one, 10(3), e0121468. ]
  14. ↑ 14.0 14.1 [Mangeney, M., Renard, M., Schlecht-Louf, G., Bouallaga, I., Heidmann, O., Letzelter, C., Richaud, A., Ducos, B., & Heidmann, T. (2007). Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins. Proceedings of the National Academy of Sciences of the United States of America, 104(51), 20534–20539. https://doi.org/10.1073/pnas.0707873105 ]
  15. ↑ [Liang, C. Y., Wang, L. J., Chen, C. P., Chen, L. F., Chen, Y. H., & Chen, H. (2010). GCM1 regulation of the expression of syncytin 2 and its cognate receptor MFSD2A in human placenta. Biology of reproduction, 83(3), 387-395]
  16. ↑ 16.0 16.1 [Lin, L., Xu, B., & Rote, N. S. (1999). Expression of endogenous retrovirus ERV-3 induces differentiation in BeWo, a choriocarcinoma model of human placental trophoblast. Placenta, 20(1), 109-118. ]
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