ABSTRACT Human herpesviruses 6A and 6B (HHV-6A/B) can integrate into the germline, resulting in inherited viral DNA—now proposed to be called “endogenous HHV-6A/B (eHHV-6A/B).” Present in 0.2–3% of humans, this integrated DNA is passed to offspring and may reactivate, posing health risks such as angina or lupus. To reduce confusion caused by varied terminology, researchers advocate using “eHHV-6A/B” for inherited forms and reserving “chromosomally integrated” for somatic integrations only.

Endogenous human herpesvirus 6 (eHHV-6) is integrated into the genomes of approximately 1

Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations. Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.

Borna disease virus 1 (BoDV1) is a disease-causing agent in some livestock and, as has recently been shown, in humans. What constitutes a protective immune response to BoDV1 is unclear. Previous studies found that endogenous bornavirus-like nucleoprotein elements (EBLNs) present in mammalian genomes produce piRNAs antisense to BoDV1 nucleoprotein mRNAs. As a known function of piRNAs is to restrict transposons via RNA interference, it has been hypothesized that EBLN-derived piRNAs may restrict BoDV1. Here we used EBLN knockout (KO) and other KO mice to test genetic factors potentially involved in antiviral immunity to BoDV1. In previous reports, BoDV1 replication was higher in mice deficient in interferon gamma, and we confirmed a role for this cytokine in BoDV1 restriction at 12 weeks post infection using mice lacking its receptor. We show that BoDV1 replicates to higher levels in the brain of mice without Toll-like receptor 7 (TLR7), suggesting a role for this innate immune receptor in BoDV1 immunity. In contrast, mice lacking piRNA-producing EBLNs were no more susceptible to BoDV1 infection than wild-type under the infection conditions used here. We thus expand the genetic evidence implicating specific conventional immune pathways in BoDV1 control and conclude that EBLN-derived piRNA-guided antiviral silencing, if it occurs, is relatively less impactful in intracerebral infection of neonates.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5′ single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4 + T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type–specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type–resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Transposable elements (TEs) or transposons are thought to play roles in animal physiological processes, such as germline, early embryonic, and brain development, as well as aging. However, their roles have not been systematically investigated through experimental studies. In this study, we created a catalog of genes directly involved in replication, excision, or integration of transposon-coding DNA, which we refer to as transposon DNA processing genes (TDPGs). Specifically, to bridge the gap to experimental studies, we sought potentially functional TDPGs which maintain intact open reading frames and the amino acids at their catalytic cores on the latest long-read genome assembly of Caenorhabditis elegans , VC2010. Among 52 519 TE loci, we identified 145 potentially functional TDPGs encoded in long terminal repeat elements, long interspersed nuclear elements, terminal inverted repeat elements, Helitrons, and Mavericks/Polintons. Our TDPG catalog, which contains a feasible number of genes, allows for the experimental manipulation of TE mobility in vivo , regardless of whether the TEs are autonomous or non-autonomous, thereby potentially promoting the study of the physiological functions of TE mobility.

Abstract A previous study suggested that fetal inheritance of chromosomally integrated human herpesvirus 6 (ici-HHV6) is associated with the hypertensive pregnancy disorder preeclampsia (PE). We aimed to study this question utilizing cord plasma samples (n = 1276) of the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort: 539 from a pregnancy with PE and 737 without. We studied these samples and 30 placentas from PE pregnancies by a multiplex qPCR for the DNAs of all nine human herpesviruses. To assess the population prevalence of iciHHV-6, we studied whole-genome sequencing data from blood-derived DNA of 3421 biobank subjects. Any herpes viral DNA was detected in only two (0.37%) PE and one (0.14%) control sample (OR 2.74, 95% CI 0.25–30.4). One PE sample contained iciHHV-6B and another HHV-7 DNA. The control’s DNA was of iciHHV-6B; the fetus having growth restriction and preterm birth without PE diagnosis. Placentas showed no herpesviruses. In the biobank data, 3 of 3421 subjects (0.08%) had low level HHV-6B but no iciHHV-6. While iciHHV-6 proved extremely rare, both fetuses with iciHHV-6B were growth-restricted, preterm, and from a pregnancy with maternal hypertension. Our findings suggest that human herpesviruses are not a significant cause of PE, whereas iciHHV-6 may pose some fetal risk.

Mobile genetic elements (MEs) are heritable mutagens that recursively generate structural variants (SVs). ME variants (MEVs) are difficult to genotype and integrate in statistical genetics, obscuring their impact on genome diversification and traits. We developed a tool that accurately genotypes MEVs using short-read whole-genome sequencing (WGS) and applied it to global human populations. We find unexpected population-specific MEV differences, including an Alu insertion distribution distinguishing Japanese from other populations. Integrating MEVs with expression quantitative trait loci (eQTL) maps shows that MEV classes regulate tissue-specific gene expression by shared mechanisms, including creating or attenuating enhancers and recruiting post-transcriptional regulators, supporting class-wide interpretability. MEVs more often associate with gene expression changes than SNVs, thus plausibly impacting traits. Performing genome-wide association study (GWAS) with MEVs pinpoints potential causes of disease risk, including a LINE-1 insertion associated with keloid and fasciitis. This work implicates MEVs as drivers of human divergence and disease risk.

AbstractTransposons are mobile DNA elements that encode genes for their own mobility. Whereas transposon copies accumulate on the genome during evolution, many lose their mobile activity due to mutations. Here, we focus on transposon-encoded genes that are directly involved in the replication, excision, and integration of transposon DNA, which we refer to as “transposon-mobility genes”, in theCaenorhabditis elegansgenome. Among the 62,773 copies of retro- and DNA transposons in the latest assembly of theC. elegansgenome (VC2010), we found that the complete open reading frame structure was conserved in 290 transposon-mobility genes. Critical amino acids at the catalytic core were conserved in only 145 of these 290 genes. Thus, in contrast to the huge number of transposon copies in the genome, only a limited number of transposons are autonomously mobile. We conclude that the comprehensive identification of potentially functional transposon-mobility genes in all transposon orders of a single species can provide a basis of molecular analysis for revealing the developmental, aging, and evolutionary roles of transposons.

Transposable elements (TEs) such as endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs) occupy nearly half of typical mammalian genomes. Previous studies show that these parasitic elements, especially LINEs and ERVs, provide important activities promoting host germ cell and placental development, preimplantation embryogenesis, and maintenance of pluripotent stem cells. Despite being the most numerically abundant type of TEs in the genome, the consequences of SINEs on host genome regulation are less well characterized than those of ERVs and LINEs. Interestingly, recent findings reveal that SINEs recruit the key architectural protein CTCF (CCCTC-binding factor), indicating a role of these elements for 3D genome regulation. Higher-order nuclear structures are linked with important cellular functions such as gene regulation and DNA replication. SINEs and other TEs, therefore, may mediate distinct physiological processes with benefits to the host by modulating the 3D genome.

Viral infection induces diverse cellular immune responses. Some viruses induce the production of antiviral cytokines, alterations of endogenous gene expression, and apoptosis; however, other viruses replicate without inducing such responses, enabling them to persistently infect cells. Infection by Borna disease virus type 1 (BoDV-1) can result in fatal immune-mediated encephalitis, including in humans, yet infection of cells in vitro is generally persistent. The regulatory mechanisms underlying this persistent infection remain unclear. Here, we show that an enhancer of RNA-silencing, TRBP, positively regulates BoDV RNA level in human cells. Knockdown of TRBP decreased BoDV RNA levels in persistently-infected cells, whereas overexpression of TRBP increased BoDV RNA levels. To investigate the mechanism underlying this phenomenon, we performed immunoprecipitation assays and found that TRBP interacts with BoDV RNA. Furthermore, we performed cell fractionation, which revealed that persistent infection with BoDV does not alter the localization of TRBP and other RNA silencing factors in cells. Our results showed the regulation of persistent BoDV infection by RNA-silencing factors in human cells.

DCG: reagents from IDbyDNA, Inc. and Illumina, Inc.; consulting for bioMerieux, Inc. NFP has no conflict of interest to disclose.

Mammalian germ cells stem from primordial germ cells (PGCs). Although the gene regulatory network controlling the development of germ cells such as PGCs is critical for ensuring gamete integrity, substantial differences exist in this network among mammalian species, suggesting that this network has been modified during mammalian evolution. Here, we show that a hominoid-specific group of endogenous retroviruses, LTR5_Hs, discloses enhancer-like signatures in human in vitro-induced PGCs, PGC-like cells (PGCLCs). Human PGCLCs exhibit a transcriptome signature similar to that of naïve-state pluripotent cells. LTR5_Hs are epigenetically activated in both PGCLCs and naïve pluripotent cells, and the expression of genes in the vicinity of LTR5_Hs is coordinately upregulated in these cell types, contributing to the establishment of the transcriptome similarity between these cell types. LTR5_Hs are preferentially bound by transcription factors that are highly expressed in both PGCLCs and naïve pluripotent cells (KLF4, TFAP2C, NANOG, and CBFA2T2), suggesting that these transcription factors contribute to the epigenetic activation of LTR5_Hs in these cells. Comparative transcriptome analysis between humans and macaques suggests that the expression of many genes in PGCLCs and naïve pluripotent cells is upregulated by LTR5_Hs insertions in the hominoid lineage. Together, this study suggests that LTR5_Hs insertions may have finetuned the gene regulatory network shared between PGCLCs and naïve pluripotent cells and coordinately altered the gene expression in these cells during hominoid evolution.

Mobile genetic elements (MEs) are heritable mutagens that contribute to divergence between lineages by recursively generating structural variants. ME variants (MEVs) are difficult to genotype, obscuring their impact on recent genome and trait diversification. We developed a tool that uses short-read sequence data to accurately genotype MEVs, enabling us to study them using statistical genetics methods in global human genomes. We observe population-specific differences in the distribution of Alu insertions that distinguish Japanese from other populations. We integrated MEVs with epigenomic and expression quantitative trait loci (eQTL) maps to determine how they impact traits. This reveals coherent patterns by which specific MEs regulate tissue-specific gene expression, including creating or attenuating enhancers and recruiting post-transcriptional regulators. We pinpoint MEVs as genetic causes of disease risk, including a LINE-1 insertion linked to keloid and other diseases of fibroblast inflammation, by introducing MEVs into the genome-wide association study (GWAS) framework. In addition to nominating previously-hidden MEVs as causes of human diseases, this work highlights MEs as accelerators of human population divergence and begins to decipher the semantics of MEs.

Gene transcription is regulated through complex mechanisms involving non-coding RNAs (ncRNAs). As the transcription of ncRNAs, especially of enhancer RNAs, is often low and cell type specific, how the levels of RNA transcription depend on genotype remains largely unexplored. Here we report the development and utility of a machine-learning model (MENTR) that reliably links genome sequence and ncRNA expression at the cell type level. Effects on ncRNA transcription predicted by the model were concordant with estimates from published studies in a cell-type-dependent manner, regardless of allele frequency and genetic linkage. Among 41,223 variants from genome-wide association studies, the model identified 7,775 enhancer RNAs and 3,548 long ncRNAs causally associated with complex traits across 348 major human primary cells and tissues, such as rare variants plausibly altering the transcription of enhancer RNAs to influence the risks of Crohn's disease and asthma. The model may aid the discovery of causal variants and the generation of testable hypotheses for biological mechanisms driving complex traits.

Acquisition of genetic material from viruses by their hosts can generate inter-host structural genome variation. We developed computational tools enabling us to study virus-derived structural variants (SVs) in population-scale whole genome sequencing (WGS) datasets and applied them to 3,332 humans. Although SVs had already been cataloged in these subjects, we found previously-overlooked virus-derived SVs. We detected non-germline SVs derived from squirrel monkey retrovirus (SMRV), human immunodeficiency virus 1 (HIV-1), and human T lymphotropic virus (HTLV-1); these variants are attributable to infection of the sequenced lymphoblastoid cell lines (LCLs) or their progenitor cells and may impact gene expression results and the biosafety of experiments using these cells. In addition, we detected new heritable SVs derived from human herpesvirus 6 (HHV-6) and human endogenous retrovirus-K (HERV-K). We report the first solo-direct repeat (DR) HHV-6 likely to reflect DR rearrangement of a known full-length endogenous HHV-6. We used linkage disequilibrium between single nucleotide variants (SNVs) and variants in reads that align to HERV-K, which often cannot be mapped uniquely using conventional short-read sequencing analysis methods, to locate previously-unknown polymorphic HERV-K loci. Some of these loci are tightly linked to trait-associated SNVs, some are in complex genome regions inaccessible by prior methods, and some contain novel HERV-K haplotypes likely derived from gene conversion from an unknown source or introgression. These tools and results broaden our perspective on the coevolution between viruses and humans, including ongoing virus-to-human gene transfer contributing to genetic variation between humans.

Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse, and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes that characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores the memory of previous exposure. Our protocol can infer CRISPR-targeted sequences, including viruses, plasmids, and previously uncharacterized elements, and predict their hosts using unassembled short-read metagenomic sequencing data. By analyzing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences, which are likely complete circular genomes. The sequences included 2,154 tailed-phage genomes, together with 257 complete crAssphage genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 56 genomes of Inoviridae species, and 95 previously uncharacterized circular small genomes that have no reliably predicted protein-coding gene. We predicted the host(s) of approximately 70% of the discovered genomes at the taxonomic level of phylum by linking protospacers to taxonomically assigned CRISPR direct repeats. These results demonstrate that our protocol is efficient for de novo inference of CRISPR-targeted sequences and their host prediction.

Understanding the genetics and taxonomy of ancient viruses will give us great insights into not only the origin and evolution of viruses but also how viral infections played roles in our evolution. Endogenous viruses are remnants of ancient viral infections and are thought to retain the genetic characteristics of viruses from ancient times. In this study, we used machine learning of endogenous RNA virus sequence signatures to identify viruses in the human genome that have not been detected or are already extinct. Here, we show that the k-mer occurrence of ancient RNA viral sequences remains similar to that of extant RNA viral sequences and can be differentiated from that of other human genome sequences. Furthermore, using this characteristic, we screened RNA viral insertions in the human reference genome and found virus-like insertions with phylogenetic and evolutionary features indicative of an exogenous origin but lacking homology to previously identified sequences. Our analysis indicates that animal genomes still contain unknown virus-derived sequences and provides a glimpse into the diversity of the ancient virosphere.

There are strong incentives for human populations to develop antiviral systems. Similarly, genomes that encode antiviral systems have had strong selective advantages. Protein-guided immune systems, which have been well studied in mammals, are necessary for survival in our virus-laden environments. Small RNA-directed antiviral immune systems suppress invasion of cells by non-self genetic material via complementary base pairing with target sequences. These RNA silencing-dependent systems operate in diverse organisms. In mammals, there is strong evidence that microRNAs (miRNAs) regulate endogenous genes important for antiviral immunity, and emerging evidence that virus-derived nucleic acids can be directly targeted by small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNAs (tRNAs) for protection in some contexts. In this review, we summarize current knowledge of the antiviral functions of each of these small RNA types and consider their conceptual and mechanistic overlap with innate and adaptive protein-guided immunity, including mammalian antiviral cytokines, as well as the prokaryotic RNA-guided immune system, CRISPR. In light of recent successes in delivery of RNA for antiviral purposes, most notably for vaccination, we discuss the potential for development of small noncoding RNA-directed antiviral therapeutics and prophylactics.