The Immunological Conundrum of Endogenous Retroelements.

Our defenses against infection rely on the ability of the immune system to distinguish invading pathogens from self. This task is exceptionally challenging, if not seemingly impossible, in the case of retroviruses that have integrated almost seamlessly into the host. This review examines the limits of innate and adaptive immune responses elicited by endogenous retroviruses and other retroelements, the targets of immune recognition, and the consequences for host health and disease. Contrary to theoretical expectation, endogenous retroelements retain substantial immunogenicity, which manifests most profoundly when their epigenetic repression is compromised, contributing to autoinflammatory and autoimmune disease and age-related inflammation. Nevertheless, recent evidence suggests that regulated immune reactivity to endogenous retroelements is integral to immune system development and function, underpinning cancer immunosurveillance, resistance to infection, and responses to the microbiota. Elucidation of the interaction points with endogenous retroelements will therefore deepen our understanding of immune system function and contribution to disease.

Human T Cell Leukemia Virus Type 1: Persistence and Pathogenesis.

Human T cell leukemia virus type 1 (HTLV-1), also known as human T lymphotropic virus type 1, was the first exogenous human retrovirus discovered. Unlike the distantly related lentivirus HIV-1, HTLV-1 causes disease in only 5-10% of infected people, depending on their ethnic origin. But whereas HIV-1 infection and the consequent diseases can be efficiently contained in most cases by antiretroviral drug treatment, there is no satisfactory treatment for the malignant or inflammatory diseases caused by HTLV-1. The purpose of the present article is to review recent advances in the understanding of the mechanisms by which the virus persists in vivo and causes disabling or fatal diseases.

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.

Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

A retroviral link to vertebrate myelination through retrotransposon-RNA-mediated control of myelin gene expression.

Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.

Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota.

The microbiota plays a fundamental role in regulating host immunity. However, the processes involved in the initiation and regulation of immunity to the microbiota remain largely unknown. Here, we show that the skin microbiota promotes the discrete expression of defined endogenous retroviruses (ERVs). Keratinocyte-intrinsic responses to ERVs depended on cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING) signaling and promoted the induction of commensal-specific T cells. Inhibition of ERV reverse transcription significantly impacted these responses, resulting in impaired immunity to the microbiota and its associated tissue repair function. Conversely, a lipid-enriched diet primed the skin for heightened ERV- expression in response to commensal colonization, leading to increased immune responses and tissue inflammation. Together, our results support the idea that the host may have co-opted its endogenous virome as a means to communicate with the exogenous microbiota, resulting in a multi-kingdom dialog that controls both tissue homeostasis and inflammation.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation.

T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.

The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer.

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.

The Role of Retrotransposons and Endogenous Retroviruses in Age-Dependent Neurodegenerative Disorders.

Over 40% of the human genome is composed of retrotransposons, DNA species that hold the potential to replicate via an RNA intermediate and are evolutionarily related to retroviruses. Retrotransposons are most studied for their ability to jump within a genome, which can cause DNA damage and novel insertional mutations. Retrotransposon-encoded products, including viral-like proteins, double-stranded RNAs, and extrachromosomal circular DNAs, can also be potent activators of the innate immune system. A growing body of evidence suggests that retrotransposons are activated in age-related neurodegenerative disorders and that such activation causally contributes to neurotoxicity. Here we provide an overview of retrotransposon biology and outline evidence of retrotransposon activation in age-related neurodegenerative disorders, with an emphasis on those involving TAR-DNA binding protein-43 (TDP-43) and tau. Studies to date provide the basis for ongoing clinical trials and hold promise for innovative strategies to ameliorate the adverse effects of retrotransposon dysregulation in neurodegenerative disorders.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

HIV integration in the human brain is linked to microglial activation and 3D genome remodeling.

To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

The developmental control of transposable elements and the evolution of higher species.

Transposable elements (TEs) account for at least 50% of the human genome. They constitute essential motors of evolution through their ability to modify genomic architecture, mutate genes and regulate gene expression. Accordingly, TEs are subject to tight epigenetic control during the earliest phases of embryonic development via histone and DNA methylation. Key to this process is recognition by sequence-specific RNA- and protein-based repressors. Collectively, these mediators are responsible for silencing a very broad range of TEs in an evolutionarily dynamic fashion. As a consequence, mobile elements and their controllers exert a marked influence on transcriptional networks in embryonic stem cells and a variety of adult tissues. The emerging picture is not that of a simple arms race but rather of a massive and sophisticated enterprise of TE domestication for the evolutionary benefit of the host.

The Lupus Susceptibility Locus Sgp3 Encodes the Suppressor of Endogenous Retrovirus Expression SNERV.

Elevated endogenous retrovirus (ERV) transcription and anti-ERV antibody reactivity are implicated in lupus pathogenesis. Overproduction of non-ecotropic ERV (NEERV) envelope glycoprotein gp70 and resultant nephritis occur in lupus-prone mice, but whether NEERV mis-expression contributes to lupus etiology is unclear. Here we identified suppressor of NEERV (Snerv) 1 and 2, Krüppel-associated box zinc-finger proteins (KRAB-ZFPs) that repressed NEERV by binding the NEERV long terminal repeat to recruit the transcriptional regulator KAP1. Germline Snerv1/Snerv2 deletion increased activating chromatin modifications, transcription, and gp70 expression from NEERV loci. F1 crosses of lupus-prone New Zealand Black (NZB) and 129 mice to Snerv1/Snerv2-/- mice failed to restore NEERV repression, demonstrating that loss of SNERV underlies the lupus autoantigen gp70 overproduction that promotes nephritis in susceptible mice and that SNERV encodes for Sgp3 (in NZB mice) and Gv-1 loci (in 129 mice). Increased ERV expression in lupus patients inversely correlated with three putative ERV-suppressing KRAB-ZFPs, suggesting that loss of KRAB-ZFP-mediated ERV control may contribute to human lupus pathogenesis.

Antibodies against endogenous retroviruses.

The human genome harbors hundreds of thousands of integrations of ancient retroviruses, amassed over millions of years of evolution. To reduce further amplification in the genome, the host prevents transcription of these now endogenous retroviruses (ERVs) through epigenetic repression and, with evolutionary time, ERVs are incapacitated by accumulating mutations and deletions. However, several members of recently endogenized ERV groups still retain the capacity to produce viral RNA, retroviral proteins, and higher order structures, including virions. The retention of viral characteristics, combined with the reversible nature of epigenetic repression, particularly as seen in cancer, allow for immunologically unanticipated ERV expression, perceived by the adaptive immune system as a genuine retroviral infection, to which it has to respond. Accordingly, antibodies reactive with ERV antigens have been detected in diverse disorders and, occasionally, in healthy individuals. Although they are part of self, the retroviral legacy of ERV antigens, and association with and, possibly, causation of disease states may set them apart from typical self-antigens. Consequently, the pathogenic or, indeed, host-protective capacity of antibodies targeting ERV antigens is likely to be context-dependent. Here, we review the immunogenicity of typical ERV proteins, with emphasis on the antibody response and its potential disease implications.

Wildlife endogenous retroviruses: colonization, consequences, and cooption.

Endogenous retroviruses (ERVs) are inherited genomic remains of past germline retroviral infections. Research on human ERVs has focused on medical implications of their dysregulation on various diseases. However, recent studies incorporating wildlife are yielding remarkable perspectives on long-term retrovirus-host interactions. These initial forays into broader taxonomic analysis, including sequencing of multiple individuals per species, show the incredible plasticity and variation of ERVs within and among wildlife species. This demonstrates that stochastic processes govern much of the vertebrate genome. In this review, we elaborate on discoveries pertaining to wildlife ERV origins and evolution, genome colonization, and consequences for host biology.

Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.

A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea.

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization's occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered. The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace's Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents (Melomys) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization.

Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development.

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.