Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells.

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture

Custom human endogenous retroviruses dedicated microarray identifies self-induced HERV-W family elements reactivated in testicular cancer upon methylation control

Endogenous retroviruses (ERVs) are an inherited part of the eukaryotic genomes, and represent ∼400 000 loci in the human genome. Human endogenous retroviruses (HERVs) can be divided into distinct families, composed of phylogenetically related but structurally heterogeneous elements. The majority of HERVs are silent in most physiological contexts, whereas a significant expression is observed in pathological contexts, such as cancers. Owing to their repetitive nature, few of the active HERV elements have been accurately identified. In addition, there are no criteria defining the active promoters among HERV long-terminal repeats (LTRs). Hence, it is difficult to understand the HERV (de)regulation mechanisms and their implication on the physiopathology of the host. We developed a microarray to specifically detect the LTR-containing transcripts from the HERV-H, HERV-E, HERV-W and HERV-K(HML-2) families. HERV transcriptome was analyzed in the placenta and seven normal/tumoral match-pair samples. We identified six HERV-W loci overexpressed in testicular cancer, including a usually placenta-restricted transcript of ERVWE1. For each locus, specific overexpression was confirmed by quantitative RT-PCR, and comparison of the activity of U3 versus U5 regions suggested a U3-promoted transcription coupled with 5′R initiation. The analysis of DNA from tumoral versus normal tissue revealed that hypomethylation of U3 promoters in tumors is a prerequisite for their activation

An endogenous lentivirus in the germline of a rodent

Lentiviruses (genus Lentivirus) are complex retroviruses that infect a broad range of mammals, including humans. Unlike many other retrovirus genera, lentiviruses have only rarely been incorporated into the mammalian germline. However, a small number of endogenous retrovirus (ERV) lineages have been identified, and these rare genomic “fossils” can provide crucial insights into the long-term history of lentivirus evolution. Here, we describe a previously unreported endogenous lentivirus lineage in the genome of the South African springhare (Pedetes capensis), demonstrating that the host range of lentiviruses has historically extended to rodents (order Rodentia). Furthermore, through comparative and phylogenetic analysis of lentivirus and ERV genomes, considering the biogeographic and ecological characteristics of host species, we reveal broader insights into the long-term evolutionary history of the genus

Paleovirological Analyses of Endogenous Retroviruses and Host Innate Immune Effectors

About 8 and 10 percent of the human and mouse genomes, respectively, are comprised of sequences of retroviral origin. Occasional infection of germ line can lead to integrated retroviral genomes being vertically inherited as host alleles. During thousands to millions of years, some of these sequences acquired inactivating mutations and were fixed in ancestral populations by genetic drift, while others became fixed by providing an evolutionary advantage to the host. Those inherited proviruses are termed endogenous retroviruses (ERVs) and have been identified in a variety of animal species representing an extensive viral “fossil” record of past retroviral infections. With the advent of whole genome sequencing projects and high throughput sequencing platforms, it became evident the wide diversity and the important role that these sequences have had in the evolution of their hosts. In the present study we developed a computational framework to identify ERVs in primate and murine genomes. The results of these genome screenings were used to identify suitable candidate sequences in which to perform paleovirological analyses that lead to the successful reconstruction of two ancient retroviruses. MuERV-L is an env-deficient highly abundant mouse specific ERV that has undergone two amplification bursts, being the more recent and prolific ~2 million years ago (MYA), probably through entirely intracellular mechanisms. MuERV-L is transcriptionally active at the two-cell stage of the mouse embryo and recent studies have implicated the co-option of its LTR as a promoter for totipotency genes. In the present work, we describe the analysis and reconstruction of an infectious ancestral MuERV-L (ancML) sequence through paleovirological analyses of MuERV-L elements in the mouse genome. The resulting ancML sequence was infectious in CHO cells and its replication was dependent on reverse transcription. We found that IFN-α could reduce ancML replication by ~20 fold. Additionally, we found that the expression of mouse APOBEC3 was able to restrict the replication of ancML. However, inspection of endogenous MuERV-L sequences suggested that the impact of APOBEC3 mediated hypermutation on MuERV-L evolution was limited. We discussed the possibility that type I IFN responses (maybe through restriction factors) might inhibit MuERV-L replication at the two-cell stage of the mouse embryo and have kept MuERV-L copy numbers under control. Although no extant human gammaretroviruses have been identified, HERV-T is a low copy primate ERV lineage that is closely related to the gammaretrovirus genus. Through phylogenetic and genomic analysis of HERV-T insertions we defined three distinct lineages. Two lineages (HERV-T1 and HERV-T2) entered the primate germline after the Old World monkey-ape split about ~32-30 MYA, whereas the other (HERV-T3) entered before this divergence ~40 MYA. Phylogenetic analysis of complete (LTR-gag-pol-env-LTR) proviral sequences showed that HERV-T2 was subjected to APOBEC3 mediated hypermutation, and subsequently expanded in apes, most likely through retrotransposon-like mechanisms. Phylogenetic and statistical analysis of HERV-T3 proviruses allowed us to estimate the sequence of their ~32 MY old ancestor, revealing that its unusually long leader sequence encoded a 855-nucleotide ORF separated from gag by 36 nucleotides. This pre-gag ORF of unknown function putatively codes for a protein that includes a transmembrane domain. Additional analysis of the HERV-T3 ancestral sequence allowed us to reconstruct the corresponding env sequence (ancHTenv). We found that a modern gammaretrovirus (MLV) could be pseudotyped with ancHTenv enabling it to infect a wide variety of primate cell lines with titers that are similar to MLV particles carrying the amphotropic MLV envelope. A single HERV-T proviral insertion in the genome of all great apes contains an env gene with full coding potential. Proteins encoded by the extant human HERV-T envelope gene (HsaHTenv) and one estimated to be encoded by the hominid ancestor were not able to generate infectious MLV pseudotyped particles, probably because HsaHTenv is not correctly processed into its mature and functional form. Statistical and phylogenetic analyses indicate that the env gene in this locus is evolving slower than the rest of the proviral sequences, and that selective pressures have acted on this locus to conserve its envelope sequence. Remarkably, we found that expression of the HsaHTenv was able to specifically block infection by MLV particles pseudotyped with the ancHTenv, but not particles pseudotyped with the amphotropic MLV envelope. Additionally, we identified MOT1 as the receptor used by ancHTenv. Further experiments are needed in order to test the hypothesis that HsaHTenv served as a restriction factor through interference with the receptor once used by HERV-T. As paleovirology also studies the evolution of the host defense mechanisms that have been shaped by past retroviral infections, we investigated the origins and evolution of tetherin, an orphan antiviral protein with no known homologs. We found that tetherin function is encoded by genes that exhibit no sequence homology and share only a common architecture and location in modern jawed vertebrate genomes, indicating an origin of ~450 MYA. Moreover, tetherin is part of a cluster of three potential sister genes that includes pv1 and a putative gene of unknown function, here referred as tm-cc(at), which encode proteins of similar architecture. Some variants of these proteins exhibit antiviral activity while others can be endowed with antiviral activity following a simple modification. Only in a slowly evolving species (coelacanths) does Tetherin exhibit homology to TMCC( aT). We suggest that neofunctionalization, drift and positive selection drove a near complete loss of sequence similarity among modern tetherin genes, and between tetherin and its sister genes. Scenarios by which this orphan gene may have arisen and evolved exemplify how protein modularity, evolvability and robustness can create new functions and preserve them, despite sequence divergence due to genetic conflict with past and present viruses

Identification and Analysis of the Heparan Sulfate-Binding Domain and Cellular Factors Involved in the Entry of Human Endogenous Retrovirus K HERV-K (HML-2)

The human endogenous retroviruses (HERVs) are remnant elements of ancient retroviruses that infected the human ancestors' germline and integrated into their genome, and thereby passed down to their descendants in a mendelian fashion. The HERV-K (HML-2) family includes the most intact and complete sequences of human-specific elements with conserved ORF for most of their proteins and the ability to produce viral particles. The viral envelope protein (Env) promoted the initial endogenization activities and was shown to have a broad tropism. It mediates the viral entry through interaction with the cell surface heparan sulfates (HS). In this study, we demonstrated first that the specified receptor binding site (RBS) of HML-2 Env is not involved in binding HS. Further, to identify the involved domain in binding HS, we aligned the protein sequence of HML-2 Env to that of the mouse mammary tumor virus (MMTV). This way, we identified an HS-binding domain (HBD) at the N-terminus of the HML-2 Env between residues 216 and 236, corresponding to MMTV HBD. Generated mutations in all positively charged residues of this domain impaired binding to heparin-coated beads and blocked viral entry. Moreover, mutations in the residues R216, K219, and K223, were influential in HS binding and the viral entry. In the second and third parts of the study, we aimed to identify the cellular factors or requirements for HML-2 Env in its early entry pathway utilizing two approaches. In the first approach, we successfully generated a trimer fusion protein for HML-2 consisting of the SU subunit fused with fibritin, a trimerization domain derived from the bacteriophage T4. The trimer fusion protein bound HS similarly to the native Env. However, it did not block HML-2 Env viral entry. Using this trimer fusion protein, we specifically co-purified the Golgi membrane protein 73 (GP73) as a potential attachment factor for HML-2 Env. The second approach included using HML-2 Env pseudotyped viral particles in conducting a functional screening of a cDNA library for transmembrane proteins in a non-permissive cell line. Screening results identified six transmembrane proteins (TMEM9, C12ORF59, IL1RAP, PSCA, LETMD1, and MPEG1) involved in the entry pathway of HML-2 that confer the susceptibility to it. We also found that only IL1RAP and MPEG1 proteins confer susceptibility in a different non-permissive cell line. Our results affirmed the role of HS in the HML-2 Env attachments and identified the specific HBD involved. They also affirmed the contribution of another molecule(s) that serves as the receptor(s). In those lines seven transmembrane proteins involved in the entry pathway of HML-2 Env were identified.Die Humanen Endogenen Retroviren (HERVs) sind Restbestandteile alter Retroviren, die die Keimbahn der menschlichen Vorfahren infizierten und in ihr Genom integriert wurden. Sie werden seither nach den Mendelschen Regeln an ihre Nachkommen weitergegeben. Die HERV-K (HML-2)-Familie umfasst die intaktesten und vollständigsten Sequenzen mit konservierten Leserahmen für die meisten ihrer Proteine und die Fähigkeit, virale Partikel zu produzieren. Das virale Hüllprotein (Env) förderte die anfängliche Endogenisierung und zeigt einen breiten Tropismus. Es vermittelt die Infektion durch erste Wechselwirkung mit Heparansulfaten (HS) auf der Zelloberfläche. In dieser Studie zeigten wir, dass die Rezeptorbindungsstelle (RBS) von HML-2 Env nicht an der Bindung von HS beteiligt ist. Um die daran beteiligte Domäne zu identifizieren, haben wir die Proteinsequenz von HML-2 Env mit der des Maus-Mammatumorvirus (MMTV) verglichen. Am N-Terminus des HML-2 Env wurde so eine potentielle homologe HS-bindende Domäne (HBD) zwischen den Positionen 216 und 236 identifiziert. Mutationen in allen positiv geladenen Resten dieser Domäne beeinträchtigten die Bindung an Heparin-beschichtete Beads und blockierten die Infektion. Darüber hinaus waren Mutationen an den Positionen R216, K219 und K223 für die HS-Bindung und den viralen Zelleintritt maßgeblich. Im zweiten und dritten Teil der Studie wollten wir die zellulären Faktoren für den HML-2 Env vermittelten Zelleintritt mit zwei Ansätzen identifizieren. Im ersten Ansatz ist es gelungen, ein Trimer-Fusionsprotein für HML-2 zu erzeugen, das aus der SU-Untereinheit besteht, die mit der Trimerisierungs-domäne fusioniert ist. Sie ist vom Fibritin des Bakteriophagen T4 abgeleitet. Das Trimer-Fusionsprotein bindet HS ähnlich wie das native Env. Es blockierte jedoch nicht den Eintritt von HML-2 in die Zellen. Mit diesem Fusionsprotein wurde das Golgi-Membranprotein 37 (GP73) als potenzieller Bindungsfaktor für HML-2 Env gefunden. Der zweite Ansatz umfasste die Verwendung von HML-2 Env-Pseudoviren bei der Durchführung eines funktionellen Screenings einer cDNA-Bibliothek mit einer nicht-permissiven Zelllinie. Damit wurden 6 Transmembranproteine (TMEM9, C12ORF59, IL1RAP, PSCA, LETMD1 und MPEG1) identifiziert, die am Infektionsprozess von HML-2 beteiligt sind. Nur IL1RAP und MPEG1 konnten allerding den Eintritt in eine andere nicht-permissiven Zelllinie vermitteln. Die Arbeiten bestätigen die Rolle von HS bei der Zelladhärenz von HML-2 Env und identifizierten die spezifische HBD. Sie bestätigten auch die Beteiligung eines oder mehrerer anderer Moleküle, die als Rezeptor(en) dienen. In diesem Zusammenhang wurden 7 Transmembranproteine identifiziert, die am Eintrittsweg von HML-2 beteiligt sind

Investigation of Endogenous Retroviruses in the Pathogenesis of Sporadic Amyotrophic Lateral Sclerosis (ALS) by

Human Endogenous Retroviruses (HERVs) are remnants of ancient retroviral infections that have become incorporated into the human genome over the course of our evolution as a species. These fossil viruses have been co-opted by our genome as regulators of cellular gene expression amongst various other functions. Over the past few years however they have been increasingly discovered as differentially expressed in neurological conditions such as Amyotrophic Lateral Sclerosis. While HERV-K (HML-2) transcripts were reported as elevated in premotor cortex samples from an ALS American cohort by Li et.al. (2015) the RT-qPCR assays performed in this sudy, using the same primers and reaction conditions in a larger ALS UK cohort were unable to corroborate these findings. Our collaborators at Kings College London, however, were able to find a novel HERV-K3 (HML-6) transcript on locus 3p21.31c upregulated in the primary motor cortex. We were able to confirm this using a subset of their primary motor cortex samples using RT-qPCR primers but were unable to replicate the results in a larger premotor cortex cohort. Using the modified ERVMap RNA-Seq method used by Jones et.al. (2021) we were able to analyse a number of publicly available datasets covering Cerebellum, Frontal Cortex, Motor Cortex and Peripheral Blood Mononuclear Cells. Within these datasets a number of novel HERVs were identified as being differentially expressed across the tissue types. A single HERV-H transcript was seen to be significantly downregulated in both the frontal cortex by RNA-Seq analysis and in the premotor cortex of our ALS UK cohort. This identification of a novel HERV-H transcript being differentially regulated in the premotor cortex and subsequent RNA-Seq analysis on the blood and the brain provides a solid basis for future research into HERVs as novel biomarkers for ALS in which a diagnostic marker for early diagnosis and target for treatment is lacking to-date

Biology of the envelope glycoproteins of sheep betaretroviruses

Retroviruses possess several biological features that differentiate them from all other infectious agents. The obligatory integration step of the retrovirus genome into the host genome has allowed these viruses to associate, modulate and alter the biology of the cell with a variety of unique mechanisms. Integration of retroviruses into the germ line of the host results in the formation of vertically transmitted “endogenous” retroviruses (ERVs). It is now becoming apparent that ERVs have often been selected as they provided evolutionary advantages to the host. Sheep Betaretroviruses provide a unique biological system to study the complex interaction between retroviruses and their hosts. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a naturally occurring lung cancer of sheep. The JSRV Env glycoprotein is a dominant oncoprotein and its expression is sufficient to induce cell transformation in vitro and in vivo. Thus, OPA is a unique large animal model for the study of lung carcinogenesis. The sheep genome harbours at least 27 copies of ERVs highly related to JSRV (enJSRVs). Studies on enJSRVs have provided evidence supporting the idea that ERVs, exogenous retroviruses and the host have coevolved through a dynamic process throughout evolution. enJSRVs play a critical role in conceptus development and placental morphogenesis, and can block JSRV replication in vitro at both early and late stages of the replication cycle. The work presented here focuses on the study of the exogenous and endogenous JSRV Envs and their role in cell transformation and trophoblast differentiation respectively. We were able to show that: I) the JSRV Env transforms epithelial cells in vitro independently from its cellular receptor; II) both the exogenous and endogenous JSRV Envs interact with the receptor tyrosine kinase RON and that the cytoplasmic tail of the Env is the major determinant modulating the biological effects of the Env-RON interaction; III) the molecular chaperone Hsp90 regulates JSRV Env induced cell transformation, in part by downregulating Akt; and IV) OPA is a useful large animal model for the evaluation of new anti-cancer therapeutic agents. Moreover, we characterized the transforming properties, receptor usage and fusogenic activity of enJSRVs Envs to gain insight into their role in placental morphogenesis. The studies described in this thesis contributed to the understanding of JSRV induced cell transformation and the biology of enJSRVs

Assembly and Compositional Analysis of Human Genomic DNA - Doctoral Dissertation, August 2002

In 1990, the United States Human Genome Project was initiated as a fifteen-year endeavor to sequence the approximately three billion bases making up the human genome (Vaughan, 1996).As of December 31, 2001, the public sequencing efforts have sequenced a total of 2.01 billion finished bases representing 63.0% of the human genome (http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsProgress.shtml&&ORG=Hs) to a Bermuda quality error rate of 1/10000 (Smith and Carrano, 1996). In addition, 1.11 billion bases representing 34.8% of the human genome has been sequenced to a rough-draft level. Efforts such as UCSC\u27s GoldenPath (Kent and Haussler, 2001) and NCBI\u27s contig assembly (Jang et al., 1999) attempt to assemble the human genome by incorporating both finished and rough-draft sequence. The availability of the human genome data allows us to ask questions concerning the maintenance of specific regions of the human genome. We consider two hypotheses for maintenance of high G+C regions: the presence of specific repetitive elements and compositional mutation biases. Our results rule out the possibility of the G+C content of repetitive elements determining regions of high and low G+C regions in the human genome. We determine that there is a compositional bias for mutation rates. However, these biases are not responsible for the maintenance of high G+C regions. In addition, we show that regions of the human under less selective pressure will mutate towards a higher A+T composition, regardless of the surrounding G+C composition. We also analyze sequence organization and show that previous studies of isochore regions (Bernardi,1993) cannot be generalized within the human genome. In addition, we propose a method to assemble only those parts of the human genome that are finished into larger contigs. Analysis of the contigs can lead to the mining of meaningful biological data that can give insights into genetic variation and evolution. I suggest a method to help aid in single nucleotide polymorphism (SNP)detection, which can help to determine differences within a population. I also discuss a dynamic-programming based approach to sequence assembly validation and detection of large-scale polymorphisms within a population that is made possible through the availability of large human sequence contigs