Gag proteins encoded by endogenous retroviruses are required for zebrafish development.

Transposable elements (TEs) make up the bulk of eukaryotic genomes and examples abound of TE-derived sequences repurposed for organismal function. The process by which TEs become coopted remains obscure because most cases involve ancient, transpositionally inactive elements. Reports of active TEs serving beneficial functions are scarce and often contentious due to difficulties in manipulating repetitive sequences. Here we show that recently active TEs in zebrafish encode products critical for embryonic development. Knockdown and rescue experiments demonstrate that the endogenous retrovirus family BHIKHARI-1 (Bik-1) encodes a Gag protein essential for mesoderm development. Mechanistically, Bik-1 Gag associates with the cell membrane and its ectopic expression in chicken embryos alters cell migration. Similarly, depletion of BHIKHARI-2 Gag, a relative of Bik-1, causes defects in neural crest development in zebrafish. We propose an "addiction" model to explain how active TEs can be integrated into conserved developmental processes.

PNMA2 forms immunogenic non-enveloped virus-like capsids associated with paraneoplastic neurological syndrome.

The paraneoplastic Ma antigen (PNMA) proteins are associated with cancer-induced paraneoplastic syndromes that present with an autoimmune response and neurological symptoms. Why PNMA proteins are associated with this severe autoimmune disease is unclear. PNMA genes are predominantly expressed in the central nervous system and are ectopically expressed in some tumors. We show that PNMA2, which has been co-opted from a Ty3 retrotransposon, encodes a protein that is released from cells as non-enveloped virus-like capsids. Recombinant PNMA2 capsids injected into mice induce autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, whereas a capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic disease show similar preferential binding to spike capsid epitopes. PNMA2 capsid-injected mice develop learning and memory deficits. These observations suggest that PNMA2 capsids act as an extracellular antigen, capable of generating an autoimmune response that results in neurological deficits.

Ribosomal profiling of human endogenous retroviruses in healthy tissues.

Human endogenous retroviruses (HERVs) are the germline embedded proviral fragments of ancient retroviral infections that make up roughly 8% of the human genome. Our understanding of HERVs in physiology primarily surrounds their non-coding functions, while their protein coding capacity remains virtually uncharacterized. Therefore, we applied the bioinformatic pipeline "hervQuant" to high-resolution ribosomal profiling of healthy tissues to provide a comprehensive overview of translationally active HERVs. We find that HERVs account for 0.1-0.4% of all translation in distinct tissue-specific profiles. Collectively, our study further supports claims that HERVs are actively translated throughout healthy tissues to provide sequences of retroviral origin to the human proteome.

HIV-1 Remission: Accelerating the Path to Permanent HIV-1 Silencing.

Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

Transposable elements: McClintock's legacy revisited.

In 1983, Barbara McClintock was awarded the Nobel Prize in Physiology or Medicine for her discovery of transposable elements. This discovery was rooted in meticulous work on maize mutants that she had carried out 40 years earlier. Over this time frame, our perception of transposable elements has undergone important paradigm shifts, with profound implications for our understanding of genome function and evolution. In commemoration of this milestone, I revisit the legacy of this iconic scientist through the kaleidoscopic history of genetics and reflect on her achievements and the hurdles she faced in her career.

Transposable elements drive the evolution of metazoan zinc finger genes.

Cys2-His2 zinc finger genes (ZNFs) form the largest family of transcription factors in metazoans. ZNF evolution is highly dynamic and characterized by the rapid expansion and contraction of numerous subfamilies across the animal phylogeny. The forces and mechanisms underlying rapid ZNF evolution remain poorly understood, but there is growing evidence that, in tetrapods, the targeting and repression of lineage-specific transposable elements (TEs) plays a critical role in the evolution of the Krüppel-associated box ZNF (KZNF) subfamily. Currently, it is unknown whether this function and coevolutionary relationship is unique to KZNFs or is a broader feature of metazoan ZNFs. Here, we present evidence that genomic conflict with TEs has been a central driver of the diversification of ZNFs in animals. Sampling from 3221 genome assemblies, we show that the copy number of retroelements correlates with that of ZNFs across at least 750 million years of metazoan evolution. Using computational predictions, we show that ZNFs preferentially bind TEs in diverse animal species. We further investigate the largest ZNF subfamily found in cyprinid fish, which is characterized by a conserved sequence we dubbed the fish N-terminal zinc finger-associated (FiNZ) domain. Zebrafish possess approximately 700 FiNZ-ZNFs, many of which are evolving adaptively under positive selection. Like mammalian KZNFs, most zebrafish FiNZ-ZNFs are expressed at the onset of zygotic genome activation, and blocking their translation using morpholinos during early embryogenesis results in derepression of transcriptionally active TEs. Together, these data suggest that ZNF diversification has been intimately connected to TE expansion throughout animal evolution.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

Rapid evolution of piRNA clusters in the Drosophila melanogaster ovary.

Animal genomes are parasitized by a horde of transposable elements (TEs) whose mutagenic activity can have catastrophic consequences. The piRNA pathway is a conserved mechanism to repress TE activity in the germline via a specialized class of small RNAs associated with effector Piwi proteins called piwi-associated RNAs (piRNAs). piRNAs are produced from discrete genomic regions called piRNA clusters (piCs). While piCs are generally enriched for TE sequences and the molecular processes by which they are transcribed and regulated are relatively well understood in Drosophila melanogaster, much less is known about the origin and evolution of piCs in this or any other species. To investigate piC evolution, we use a population genomics approach to compare piC activity and sequence composition across 8 geographically distant strains of D. melanogaster with high quality long-read genome assemblies. We perform extensive annotations of ovary piCs and TE content in each strain and test predictions of two proposed models of piC evolution. The 'de novo' model posits that individual TE insertions can spontaneously attain the status of a small piC to generate piRNAs silencing the entire TE family. The 'trap' model envisions large and evolutionary stable genomic clusters where TEs tend to accumulate and serves as a long-term "memory" of ancient TE invasions and produce a great variety of piRNAs protecting against related TEs entering the genome. It remains unclear which model best describes the evolution of piCs. Our analysis uncovers extensive variation in piC activity across strains and signatures of rapid birth and death of piCs in natural populations. Most TE families inferred to be recently or currently active show an enrichment of strain-specific insertions into large piCs, consistent with the trap model. By contrast, only a small subset of active LTR retrotransposon families is enriched for the formation of strain-specific piCs, suggesting that these families have an inherent proclivity to form de novo piCs. Thus, our findings support aspects of both 'de novo' and 'trap' models of piC evolution. We propose that these two models represent two extreme stages along an evolutionary continuum, which begins with the emergence of piCs de novo from a few specific LTR retrotransposon insertions that subsequently expand by accretion of other TE insertions during evolution to form larger 'trap' clusters. Our study shows that piCs are evolutionarily labile and that TEs themselves are the major force driving the formation and evolution of piCs.

Transposable Element Interactions Shape the Ecology of the Deer Mouse Genome.

The genomic landscape of transposable elements (TEs) varies dramatically across species, with some TEs demonstrating greater success in colonizing particular lineages than others. In mammals, long interspersed nuclear element (LINE) retrotransposons are typically more common than any other TE. Here, we report an unusual genomic landscape of TEs in the deer mouse, Peromyscus maniculatus. In contrast to other previously examined mammals, long terminal repeat elements occupy more of the deer mouse genome than LINEs (11% and 10%, respectively). This pattern reflects a combination of relatively low LINE activity and a massive invasion of lineage-specific endogenous retroviruses (ERVs). Deer mouse ERVs exhibit diverse origins spanning the retroviral phylogeny suggesting they have been host to a wide range of exogenous retroviruses. Notably, we trace the origin of one ERV lineage, which arose ∼5-18 million years ago, to a close relative of feline leukemia virus, revealing inter-ordinal horizontal transmission. Several lineage-specific ERV subfamilies have very high copy numbers, with the top five most abundant accounting for ∼2% of the genome. We also observe a massive amplification of Kruppel-associated box domain-containing zinc finger genes, which likely control ERV activity and whose expansion may have been facilitated by ectopic recombination between ERVs. Finally, we find evidence that ERVs directly impacted the evolutionary trajectory of LINEs by outcompeting them for genomic sites and frequently disrupting autonomous LINE copies. Together, our results illuminate the genomic ecology that shaped the unique deer mouse TE landscape, shedding light on the evolutionary processes that give rise to variation in mammalian genome structure.

PNMA2 forms non-enveloped virus-like capsids that trigger paraneoplastic neurological syndrome.

The paraneoplastic Ma antigen (PNMA) genes are associated with cancer-induced paraneoplastic syndromes that present with neurological symptoms and autoantibody production. How PNMA proteins trigger a severe autoimmune disease is unclear. PNMA genes are predominately expressed in the central nervous system with little known functions but are ectopically expressed in some tumors. Here, we show that PNMA2 is derived from a Ty3 retrotransposon that encodes a protein which forms virus-like capsids released from cells as non-enveloped particles. Recombinant PNMA2 capsids injected into mice induce a robust autoimmune reaction with significant generation of autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, while capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies present in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic neurologic disease show similar preferential binding to PNMA2 "spike" capsid epitopes. These observations suggest that PNMA2 capsids released from tumors trigger an autoimmune response that underlies Ma2 paraneoplastic neurological syndrome.

A comprehensive SARS-CoV-2-human protein-protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets.

Studying viral-host protein-protein interactions can facilitate the discovery of therapies for viral infection. We use high-throughput yeast two-hybrid experiments and mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of 739 high-confidence binary and co-complex interactions, validating 218 known SARS-CoV-2 host factors and revealing 361 novel ones. Our results show the highest overlap of interaction partners between published datasets and of genes differentially expressed in samples from COVID-19 patients. We identify an interaction between the viral protein ORF3a and the human transcription factor ZNF579, illustrating a direct viral impact on host transcription. We perform network-based screens of >2,900 FDA-approved or investigational drugs and identify 23 with significant network proximity to SARS-CoV-2 host factors. One of these drugs, carvedilol, shows clinical benefits for COVID-19 patients in an electronic health records analysis and antiviral properties in a human lung cell line infected with SARS-CoV-2. Our study demonstrates the value of network systems biology to understand human-virus interactions and provides hits for further research on COVID-19 therapeutics.

Transposable elements may enhance antiviral resistance in HIV-1 elite controllers.

Less than 0.5% of people living with HIV-1 are elite controllers (ECs) - individuals who have a replication-competent viral reservoir in their CD4+ T cells but maintain undetectable plasma viremia without the help of antiretroviral therapy. While the EC CD4+ T cell transcriptome has been investigated for gene expression signatures associated with disease progression (or, in this case, a lack thereof), the expression and regulatory activity of transposable elements (TEs) in ECs has not been explored. Yet previous studies have established that TEs can directly impact the immune response to pathogens, including HIV-1. Thus, we hypothesize that the regulatory activities of TEs could contribute to the natural resistance of ECs against HIV-1. We perform a TE-centric analysis of previously published multi-omics data derived from EC individuals and other populations. We find that the CD4+ T cell transcriptome and retrotranscriptome of ECs are distinct from healthy controls, treated patients, and viremic progressors. However, there is a substantial level of transcriptomic heterogeneity among ECs. We categorize individuals with distinct chromatin accessibility and expression profiles into four clusters within the EC group, each possessing unique repertoires of TEs and antiviral factors. Notably, several TE families with known immuno-regulatory activity are differentially expressed among ECs. Their transcript levels in ECs positively correlate with their chromatin accessibility and negatively correlate with the expression of their KRAB zinc-finger (KZNF) repressors. This coordinated variation is seen at the level of individual TE loci likely acting or, in some cases, known to act as cis-regulatory elements for nearby genes involved in the immune response and HIV-1 restriction. Based on these results, we propose that the EC phenotype is driven in part by the reduced availability of specific KZNF proteins to repress TE-derived cis-regulatory elements for antiviral genes, thereby heightening their basal level of resistance to HIV-1 infection. Our study reveals considerable heterogeneity in the CD4+ T cell transcriptome of ECs, including variable expression of TEs and their KZNF controllers, that must be taken into consideration to decipher the mechanisms enabling HIV-1 control.

Transposon control as a checkpoint for tissue regeneration.

Tissue regeneration requires precise temporal control of cellular processes such as inflammatory signaling, chromatin remodeling and proliferation. The combination of these processes forms a unique microenvironment permissive to the expression, and potential mobilization of, transposable elements (TEs). Here, we develop the hypothesis that TE activation creates a barrier to tissue repair that must be overcome to achieve successful regeneration. We discuss how uncontrolled TE activity may impede tissue restoration and review mechanisms by which TE activity may be controlled during regeneration. We posit that the diversification and co-evolution of TEs and host control mechanisms may contribute to the wide variation in regenerative competency across tissues and species.

Evolution and antiviral activity of a human protein of retroviral origin.

Endogenous retroviruses are abundant components of mammalian genomes descended from ancient germline infections. In several mammals, the envelope proteins encoded by these elements protect against exogenous viruses, but this activity has not been documented with endogenously expressed envelopes in humans. We report that the human genome harbors a large pool of envelope-derived sequences with the potential to restrict retroviral infection. To test this, we characterized an envelope-derived protein, Suppressyn. We found that Suppressyn is expressed in human preimplantation embryos and developing placenta using its ancestral retroviral promoter. Cell culture assays showed that Suppressyn, and its hominoid orthologs, could restrict infection by extant mammalian type D retroviruses. Our data support a generalizable model of retroviral envelope co-option for host immunity and genome defense.

A comprehensive SARS-CoV-2-human protein-protein interactome network identifies pathobiology and host-targeting therapies for COVID-19.

Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host's immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions. We report a total of 739 high-confidence interactions, showing the highest overlap of interaction partners among published datasets as well as the highest overlap with genes differentially expressed in samples (such as upper airway and bronchial epithelial cells) from patients with SARS-CoV-2 infection. Showcasing the utility of our network, we describe a novel interaction between the viral accessory protein ORF3a and the host zinc finger transcription factor ZNF579 to illustrate a SARS-CoV-2 factor mediating a direct impact on host transcription. Leveraging our interactome, we performed network-based drug screens for over 2,900 FDA-approved/investigational drugs and obtained a curated list of 23 drugs that had significant network proximities to SARS-CoV-2 host factors, one of which, carvedilol, showed promising antiviral properties. We performed electronic health record-based validation using two independent large-scale, longitudinal COVID-19 patient databases and found that carvedilol usage was associated with a significantly lowered probability (17%-20%, P < 0.001) of obtaining a SARS-CoV-2 positive test after adjusting various confounding factors. Carvedilol additionally showed anti-viral activity against SARS-CoV-2 in a human lung epithelial cell line [half maximal effective concentration (EC 50 ) value of 4.1 µM], suggesting a mechanism for its beneficial effect in COVID-19. Our study demonstrates the value of large-scale network systems biology approaches for extracting biological insight from complex biological processes.

Roles of transposable elements in the regulation of mammalian transcription.

Transposable elements (TEs) comprise about half of the mammalian genome. TEs often contain sequences capable of recruiting the host transcription machinery, which they use to express their own products and promote transposition. However, the regulatory sequences carried by TEs may affect host transcription long after the TEs have lost the ability to transpose. Recent advances in genome analysis and engineering have facilitated systematic interrogation of the regulatory activities of TEs. In this Review, we discuss diverse mechanisms by which TEs contribute to transcription regulation. Notably, TEs can donate enhancer and promoter sequences that influence the expression of host genes, modify 3D chromatin architecture and give rise to novel regulatory genes, including non-coding RNAs and transcription factors. We discuss how TEs spur regulatory evolution and facilitate the emergence of genetic novelties in mammalian physiology and development. By virtue of their repetitive and interspersed nature, TEs offer unique opportunities to dissect the effects of mutation and genomic context on the function and evolution of cis-regulatory elements. We argue that TE-centric studies hold the key to unlocking general principles of transcription regulation and evolution.

Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo.

The human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least eight previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by a mixture of mutational processes, including point mutations, duplications, and multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo.

Single-Cell Analysis Reveals Unexpected Cellular Changes and Transposon Expression Signatures in the Colonic Epithelium of Treatment-Naïve Adult Crohn's Disease Patients.

BACKGROUND & AIMS: The intestinal barrier comprises a monolayer of specialized intestinal epithelial cells (IECs) that are critical in maintaining mucosal homeostasis. Dysfunction within various IEC fractions can alter intestinal permeability in a genetically susceptible host, resulting in a chronic and debilitating condition known as Crohn's disease (CD). Defining the molecular changes in each IEC type in CD will contribute to an improved understanding of the pathogenic processes and the identification of cell type-specific therapeutic targets. We performed, at single-cell resolution, a direct comparison of the colonic epithelial cellular and molecular landscape between treatment-naïve adult CD and non-inflammatory bowel disease control patients. METHODS: Colonic epithelial-enriched, single-cell sequencing from treatment-naïve adult CD and non-inflammatory bowel disease patients was investigated to identify disease-induced differences in IEC types. RESULTS: Our analysis showed that in CD patients there is a significant skew in the colonic epithelial cellular distribution away from canonical LGR5+ stem cells, located at the crypt bottom, and toward one specific subtype of mature colonocytes, located at the crypt top. Further analysis showed unique changes to gene expression programs in every major cell type, including a previously undescribed suppression in CD of most enteroendocrine driver genes as well as L-cell markers including GCG. We also dissect an incompletely understood SPIB+ cell cluster, revealing at least 4 subclusters that likely represent different stages of a maturational trajectory. One of these SPIB+ subclusters expresses crypt-top colonocyte markers and is up-regulated significantly in CD, whereas another subcluster strongly expresses and stains positive for lysozyme (albeit no other canonical Paneth cell marker), which surprisingly is greatly reduced in expression in CD. In addition, we also discovered transposable element markers of colonic epithelial cell types as well as transposable element families that are altered significantly in CD in a cell type-specific manner. Finally, through integration with data from genome-wide association studies, we show that genes implicated in CD risk show heretofore unknown cell type-specific patterns of aberrant expression in CD, providing unprecedented insight into the potential biological functions of these genes. CONCLUSIONS: Single-cell analysis shows a number of unexpected cellular and molecular features, including transposable element expression signatures, in the colonic epithelium of treatment-naïve adult CD.