Yuki Nakaya

Identification of Novel Endogenous Betaretroviruses Which Are Transcribed in the Bovine Placenta

ABSTRACT Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3′ long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.

Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation

ABSTRACT During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin-1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution.

Dynamic Evolution of Endogenous Retrovirus-Derived Genes Expressed in Bovine Conceptuses during the Period of Placentation

In evolution of mammals, some of essential genes for placental development are known to be of retroviral origin, as syncytin-1 derived from an envelope (env) gene of an endogenous retrovirus (ERV) aids in the cell fusion of placenta in humans. Although the placenta serves the same function in all placental mammals, env-derived genes responsible for trophoblast cell fusion and maternal immune tolerance differ among species and remain largely unidentified in the bovine species. To examine env-derived genes playing a role in the bovine placental development comprehensively, we determined the transcriptomic profiles of bovine conceptuses during three crucial windows of implantation periods using a high-throughput sequencer. The sequence reads were mapped into the bovine genome, in which ERV candidates were annotated using RetroTector© (7,624 and 1,542 for ERV-derived and env-derived genes, respectively). The mapped reads showed that approximately 18% (284 genes) of env-derived genes in the genome were expressed during placenta formation, and approximately 4% (63 genes) were detected for all days examined. We verified three env-derived genes that are expressed in trophoblast cells by polymerase chain reaction. Out of these three, the sequence of env-derived gene with the longest open reading frame (named BERV-P env) was found to show high expression levels in trophoblast cell lines and to be similar to those of syncytin-Car1 genes found in dogs and cats, despite their disparate origins. These results suggest that placentation depends on various retrovirus-derived genes that could have replaced endogenous predecessors during evolution.

Bovine trophoblastic cell differentiation and binucleation involves enhanced endogenous retrovirus element expression

BackgroundEndogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines.MethodsIn this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE–B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry.ResultsbERVE-A, bERVE–B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P < 0.01).ConclusionsThese results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.

Identification and Functional Analysis of Three Isoforms of Bovine BST-2

Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.

The Roles of Syncytin-Like Proteins in Ruminant Placentation

Recent developments in genome sequencing techniques have led to the identification of huge numbers of endogenous retroviruses (ERV) in various mammals. ERVs, which occupy 8%–13% of mammalian genomes, are believed to affect mammalian evolution and biological diversity. Although the functional significance of most ERVs remains to be elucidated, several ERVs are thought to have pivotal roles in host physiology. We and other groups recently identified ERV envelope proteins (e.g., Fematrin-1, Syncytin-Rum1, endogenous Jaagsiekte sheep retrovirus Env) that may determine the morphogenesis of the unique fused trophoblast cells, termed trinucleate cells and syncytial plaques, found in ruminant placentas; however, there are still a number of outstanding issues with regard to the role of ERVs that remain to be resolved. Here, we review what is known about how these ERVs have contributed to the development of ruminant-specific trophoblast cells.

Dysfunction of Bovine Endogenous Retrovirus K2 Envelope Glycoprotein Is Related to Unsuccessful Intracellular Trafficking

ABSTRACT Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. IMPORTANCE Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry receptor and virus-host membrane fusion, respectively. However, unexplained issues remain for the maturation process of retroviral Env. We previously reported that bovine endogenous retrovirus K2 (BERV-K2) Env lost fusogenicity due to a defect in the cleavage of SU and TM. In this study, we identified that mutations residing in BERV-K2 SU disturbed intracellular trafficking of BERV-K2 Env and resulted its inefficient cleavage. Because SU is not known to play an important role in this process, our study may provide novel insights into the maturation mechanism of retroviral Envs.

Epigenetic regulation on the 5'-proximal CpG island of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B.

Porcine endogenous retroviruses (PERVs) have been considered one of the major risks of xenotransplantation from pigs to humans. PERV-A efficiently utilizes human PERV-A receptor 2 (HuPAR-2)/GPR172B to infect human cells; however, there has been no study on the regulation mechanisms of HuPAR-2/GPR172B expression. In this study, we examined the expression of HuPAR-2/GPR172B from the standpoint of epigenetic regulation and discussed the risks of PERV-A infection in xenotransplantation. Quantitative real-time RT-PCR revealed that HuPAR-2 mRNA was preferentially expressed in placental tissue, whereas it was highly suppressed in BeWo cells (a human choriocarcinoma cell line) and HEK293 cells. A CpG island containing the HuPAR-2 transcription starting site was identified by in silico analysis. The DNA methylation ratio (the relative quantity of methylcytosine to total cytosine) and histone modification (H3K9me3) levels in the CpG island measured by bisulfite genomic sequencing and ChIP assay, respectively, were inversely correlated with the mRNA levels. Both HuPAR-2 mRNA and HuPAR-2 protein were up-regulated in HEK293 cells by inhibiting DNA methylation and histone deacetylation. Additionally, promoter/enhancer activities within the CpG island were suppressed by in vitro DNA methylation. Our results demonstrated that epigenetic modification regulates HuPAR-2 expression.

Mapping of a neutralizing epitope in the surface envelope protein of porcine endogenous retrovirus subgroup B.

Pigs are thought to be the most suitable donor animal for xenotransplantation. However, pigs harbour potentially hazardous infectious agents, termed porcine endogenous retroviruses (PERVs), in their genome. In this study, we generated a mAb against PERV-B surface (SU) envelope protein (Env), designated KRT1. KRT1 binding was detected by an indirect immunofluorescence assay and flow cytometric analysis on cells infected with PERV-B. KRT1 neutralized PERV-B pseudotype virus and specifically recognized PERV-B SU Env, but not PERV-A SU Env by immunoblotting analysis. The peptide-ELISA revealed that KRT1 recognized a linear peptide sequence (ALEPPHNLPVP) residing in a proline-rich region that is one of the subdomains of SU Env. In conclusion, the KRT1 antibody will serve as a useful tool for the study of PERV-B and, more importantly, it may provide new protective strategies against PERV-B infection in xenotransplantation.

Binding of transcription factor activating protein 2 γ on the 5′-proximal promoter region of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B

Nakaya Y, Shimode S, Kobayashi T, Imakawa K, Miyazawa T. Binding of transcription factor activating protein 2γ on the 5′‐proximal promoter region of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B. Xenotransplantation 2012; 19: 177–185.