Mobile element variation contributes to population-specific genome diversification, gene regulation and disease 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.

Bridging multiple dimensions: roles of transposable elements in higher-order genome regulation.

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.

The regulation of persistent Borna disease virus infection by RNA silencing factors in human cells.

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.

Prediction of the cell-type-specific transcription of non-coding RNAs from genome sequences via machine learning.

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.

A hominoid-specific endogenous retrovirus may have rewired the gene regulatory network shared between primordial germ cells and naïve pluripotent cells.

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.

Mammalian antiviral systems directed by small RNA.

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.

Comprehensive discovery of CRISPR-targeted terminally redundant sequences in the human gut metagenome: Viruses, plasmids, and more.

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.

Virus-derived variation in diverse human genomes.

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.

Virus-like insertions with sequence signatures similar to those of endogenous nonretroviral RNA viruses in the human genome.

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.

Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration out of Africa.

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or "reactivation" of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

Endogenization and excision of human herpesvirus 6 in human genomes.

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.

Prevalence and Spectrum of Pathogenic Germline Variants in Japanese Patients With Early-Onset Colorectal, Breast, and Prostate Cancer.

PUPOSE: We investigated the prevalence and spectrum of pathogenic germline variants in patients with early-onset colorectal cancer (CRC), breast cancer (BC), and prostate cancer (PCA) in the Japanese population. We also identified pathogenic variants in other cancer risk genes, giving consideration to future multigene testing panels for this population. METHODS: We performed whole-genome sequencing for 1,037 Japanese individuals, including patients with early-onset CRC (n = 196), BC (n = 237), and PCA (n = 215) and controls (n = 389). We screened for pathogenic variants, including single nucleotide variants and copy number variants, among well-established first-tier cancer genes for each cancer type and examined an expended second-tier panel including cancer-predisposing genes from the Cancer Gene Census. RESULTS: Proportions of patients with germline pathogenic variants differed by cancer subgroup, with the highest in BC (14.8%), followed by CRC (9.2%), and PCA (3.7%). In contrast, 2 of 389 control subjects (0.5%) carried a germline pathogenic variant. In comparison with controls, the proportion of patients with pathogenic variants in the second-tier panel was increased significantly for PCA (3.7% to 11.6%, P = 2.96 × 10-4), but not for CRC or BC, after multitesting adjustment. In patients with PCA, DNA repair pathway genes in the extended panel often contained pathogenic variants (P = .011). CONCLUSION: Our analyses support the clinical usefulness of established cancer gene panels in the Japanese population for 3 major cancer types. Additional genes, especially those involved in DNA repair, might be considered for developing multipanel testing in Japanese patients with early-onset PCA.

piRNA-Guided CRISPR-like Immunity in Eukaryotes.

Eukaryotic genomes contain virus-derived sequences called endogenous virus elements (EVEs). The majority of EVEs are related to retroviruses, which integrate into the host genome in order to replicate. Some retroviral EVEs encode a function; for example, some produce proteins that block infection by related viruses. EVEs derived from nonretroviral viruses - also recently found in many eukaryotic genomes - are more enigmatic. Here, we summarize the evidence that EVEs can act as templates to generate Piwi-interacting RNAs (piRNAs), whose canonical function is sequence-specific silencing of transposable elements (TEs) to maintain genomic integrity. We argue that EVEs may thus enable heritable, sequence-specific antiviral immune memory in eukaryotes - analogous to CRISPR-Cas immunity in prokaryotes.

The Changing Face of Liver Transplantation in the United States: The Effect of HCV Antiviral Eras on Transplantation Trends and Outcomes.

BACKGROUND: Hepatitis C virus (HCV) cirrhosis is the leading indication for liver transplantation in the United States, although nonalcoholic steatohepatitis (NASH) is on the rise. Increasingly effective HCV antivirals are available, but their association with diagnosis-specific liver transplantation rates and early graft survival is not known. METHODS: The Scientific Registry of Transplant Recipients database records were retrospectively stratified by HCV antiviral era: interferon (2003-2010), protease inhibitors (2011-2013), and direct-acting antivirals (2014 to present). Kaplan-Meier, χ2, and multivariable Cox proportional hazards regression models evaluated the effects of antiviral era and etiology of liver disease on transplantation rates and graft survival over 3 years. RESULTS: Liver transplants for HCV decreased (35.3% to 23.6%), whereas those for NASH and alcoholic liver disease increased (5.8% to 16.5% and 15.6% to 24.0%) with each advancing era (all P < 0.05). Early graft survival improved with each advancing era for HCV but not for hepatitis B virus, NASH, or alcoholic liver disease (multivariable model era by diagnosis interaction P < 0.001). Era-specific multivariable models demonstrated that the risk of early graft loss for NASH was 22% lower than for HCV in the interferon era (hazard ratio, 0.78; 95% confidence interval, 0.64-0.96; P = 0.02) but risks associated with these diagnoses did not differ significantly in the protease inhibitor (P = 0.06) or direct-acting antiviral eras (P = 0.08). CONCLUSIONS: Increasing effectiveness of HCV antivirals corresponds with decreased rates of liver transplantation for HCV and improved early graft survival. As the rates of liver transplant for NASH continue to increase, focus will be needed on the prevention and effective therapies for this disease.

Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.

HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.

A Viral (Arc)hive for Metazoan Memory.

Arc, a master regulator of synaptic plasticity, contains sequence elements that are evolutionarily related to retrotransposon Gag genes. Two related papers in this issue of Cell show that Arc retains retroviral-like capsid-forming ability and can transmit mRNA between cells in the nervous system, a process that may be important for synaptic function.

Endogenized viral sequences in mammals.

Reverse-transcribed RNA molecules compose a significant portion of the human genome. Many of these RNA molecules were retrovirus genomes either infecting germline cells or having done so in a previous generation but retaining transcriptional activity. This mechanism itself accounts for a quarter of the genomic sequence information of mammals for which there is data. We understand relatively little about the causes and consequences of retroviral endogenization. This review highlights functions ascribed to sequences of viral origin endogenized into mammalian genomes and suggests some of the most pressing questions raised by these observations.