The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

LTR retroelement expansion of the human cancer transcriptome and immunopeptidome revealed by de novo transcript assembly.

Dysregulated endogenous retroelements (EREs) are increasingly implicated in the initiation, progression, and immune surveillance of human cancer. However, incomplete knowledge of ERE activity limits mechanistic studies. By using pan-cancer de novo transcript assembly, we uncover the extent and complexity of ERE transcription. The current assembly doubled the number of previously annotated transcripts overlapping with long-terminal repeat (LTR) elements, several thousand of which were expressed specifically in one or a few related cancer types. Exemplified in melanoma, LTR-overlapping transcripts were highly predictable, disease prognostic, and closely linked with molecularly defined subtypes. They further showed the potential to affect disease-relevant genes, as well as produce novel cancer-specific antigenic peptides. This extended view of LTR elements provides the framework for functional validation of affected genes and targets for cancer immunotherapy.

Retrotransposon expression in response to in vitro inoculation with two fungal pathogens of Scots pine (Pinus sylvestris L.).

OBJECTIVE: Conifer genomes show high genetic diversity in intergenic regions that contain diverse sets of transposable elements with dominating long terminal repeat (LTR) retrotransposons (RE). Transcription of RE in response to environmental stimuli could produce various types of regulatory non-coding RNAs, but global genomic methylation changes could result in a coincidental expression of normally silent genomic regions. Expression of several RE families was evaluated in Scots pine seedlings after controlled inoculations with two fungal species that exhibit different modes of pathogenicity (necrotrophic and likely biotrophic); data compared to the overall RE distribution in genome. Recognition of regulatory non-coding RNA involved in host-pathogen interplay could be valuable in understanding defence mechanisms of perennial plants. RESULTS: In the case of necrotrophic fungi Heterobasidion annosum (HA), short activation followed by restriction of RE expression was revealed after inoculation and during the spread of the pathogen. After inoculation with Lophodermium seditiosum (LS), an early increase in RE expression was revealed with the spread of the pathogen and subsequent transcription rise in all seedlings. Our observations indicate that in the complex plant genome multiple RE families constitutively express in response to pathogen invasion and these sequences could undergo regulation related to host response or pathogen influence.

Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are normally suppressed in somatic tissues mainly due to DNA methylation and antiviral defense. However, the mechanism to suppress L1s may be disrupted in cancers, thus allowing L1s to act as insertional mutagens and cause genomic rearrangement and instability. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, we report multiple correlates of L1 activity in stomach, colorectal, and esophageal tumors through an integrative analysis of cancer whole-genome and matched RNA-sequencing profiles. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. A potential L1 expression suppressor, TP53, was mutated in tumors with frequent L1 insertions. We characterized the effects of somatic L1 insertions on mRNA splicing and expression, and demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found that a cancer-specific L1 insertion in an exon of MOV10, a key L1 suppressor, caused exon skipping and decreased expression of the affected allele due to nonsense-mediated decay in a tumor with a high L1 insertion load. Importantly, tumors with high immune activity, for example, those associated with Epstein-Barr virus infection or microsatellite instability, tended to carry a low number of L1 insertions in genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1 Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers.

Surveillance of Retroelement Expression and Nucleic-Acid Immunity by Histone Methyltransferase SETDB1.

In human cancers, histone methyltransferase SETDB1 (SET domain, bifurcated 1) is frequently overexpressed but its significance in carcinogenesis remains elusive. A recent study shows that SETDB1 downregulation induces de-repression of retroelements and innate immunity in cancer cells. The possibility of SETDB1 functioning as a surveillant of retroelement expression is discussed in this study: the cytoplasmic presence of retroelement-derived nucleic acids (RdNAs) drives SETDB1 into the nucleus by the RNA-interference route, rendering the corresponding retroelement transcriptionally inert. These RdNAs could, therefore, be signals of genome instability sent out for SETDB1 present in the cytoplasm to maintain genome integrity.

Epigenetic control of retrotransposons in adult tissues: implications for immune regulation.

Retrotransposons tune immune reactivity in differentiated cells because when they are transcribed, their nucleic acids can be viewed as non-self leading to innate immune sensing. Most retrotransposons, however, are subject to transcriptional regulation by a multitude of epigenetic pathways, which have coevolved with them for millions of years. While a lot is known about the epigenetic control of retrotransposons in germ cells and early embryos, surprisingly little is understood about these pathways in adult tissues, particularly in human cells. Recent evidence suggests that retrotransposon repression persists in differentiated cells and is dynamic. Future insight into this topic may teach us how to reactivate or silence specific retrotransposon families, to promote anti-tumor immunity or dampen autoimmunity through epigenetic modulation.

Immune responses to endogenous retroelements: taking the bad with the good.

The ultimate form of parasitism and evasion of host immunity is for the parasite genome to enter the germ line of the host species. Retroviruses have invaded the host germ line on the grandest scale, and this is evident in the extraordinary abundance of endogenous retroelements in the genome of all vertebrate species that have been studied. Many of these endogenous retroelements have retained viral characteristics; some also the capacity to replicate and, consequently, the potential to trigger host innate and adaptive immune responses. However, although retroelements are mainly recognized for their pathogenic potential, recent evidence suggests that this 'enemy within' may also have beneficial roles in tuning host immune reactivity. In this Review, we discuss how the immune system recognizes and is shaped by endogenous retroelements.

The enemy within: endogenous retroelements and autoimmune disease.

Inappropriate or chronic detection of self nucleic acids by the innate immune system underlies many human autoimmune diseases. We discuss here an unexpected source of endogenous immunostimulatory nucleic acids: the reverse-transcribed cDNA of endogenous retroelements. The interplay between innate immune sensing and clearance of retroelement cDNA has important implications for the understanding of immune responses to infectious retroviruses such as human immunodeficiency virus (HIV). Furthermore, the detection of cDNA by the innate immune system reveals an evolutionary tradeoff: selection for a vigorous, sensitive response to infectious retroviruses may predispose the inappropriate detection of endogenous retroelements. We propose that this tradeoff has placed unique constraints on the sensitivity of the DNA-activated antiviral response, with implications for the interactions of DNA viruses and retroviruses with their hosts. Finally, we discuss how better understanding of the intersection of retroelement biology and innate immunity can guide the way to novel therapies for specific autoimmune diseases.

Vaccination against endogenous retrotransposable element consensus sequences does not protect rhesus macaques from SIVsmE660 infection and replication.

The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.

Differential inhibition of LINE1 and LINE2 retrotransposition by vertebrate AID/APOBEC proteins.

BACKGROUND: The role of AID/APOBEC proteins in the mammalian immune response against retroviruses and retrotransposons is well established. G to A hypermutations, the hallmark of their cytidine deaminase activity, are present in several mammalian retrotransposons. However, the role of AID/APOBEC proteins in non-mammalian retroelement restriction is not completely understood. RESULTS: Here we provide the first evidence of anti-retroelement activity of a reptilian APOBEC protein. The green anole lizard A1 protein displayed potent DNA mutator activity and inhibited ex vivo retrotransposition of LINE1 and LINE2 ORF1 protein encoding elements, displaying a mechanism of action similar to that of the human A1 protein. In contrast, the human A3 proteins did not require ORF1 protein to inhibit LINE retrotransposition, suggesting a differential mechanism of anti-LINE action of A1 proteins, which emerged in amniotes, and A3 proteins, exclusive to placental mammals. In accordance, genomic analyses demonstrate differential G to A DNA editing of LINE retrotransposons in the lizard genome, which is also the first evidence for G to A DNA editing in non-mammalian genomes. CONCLUSION: Our data suggest that vertebrate APOBEC proteins differentially inhibit the retrotransposition of LINE elements and that the anti-retroelement activity of APOBEC proteins predates mammals.

Endogenous retroelements and autoimmune disease.

Innate immune sensors of foreign nucleic acids are essential for antiviral immunity, but these same sensors can cause autoimmune disease through inappropriate detection of self-nucleic acids. The sources of the endogenous RNA and DNA that trigger autoreactive responses include chromatin and ribonucleoproteins that are the targets of autoantibodies in numerous autoimmune diseases, including systemic lupus erythematosus. In this review, I discuss recent data implicating endogenous retroelements-viruses that make up a substantial fraction of our genomes-as an important source of endogenous nucleic acids that can cause autoimmune disease. Understanding this potentially pathologic role for retroelements and the precise mechanisms by which their genomes are sensed and metabolized has important implications for the diagnosis and treatment of numerous autoimmune disorders.

The breadth of antiviral activity of Apobec3DE in chimpanzees has been driven by positive selection.

The Apobec3 family of cytidine deaminases can inhibit the replication of retroviruses and retrotransposons. Human and chimpanzee genomes encode seven Apobec3 paralogs; of these, Apobec3DE has the greatest sequence divergence between humans and chimpanzees. Here we show that even though human and chimpanzee Apobec3DEs are very divergent, the two orthologs similarly restrict long terminal repeat (LTR) and non-LTR retrotransposons (MusD and Alu, respectively). However, chimpanzee Apobec3DE also potently restricts two lentiviruses, human immunodeficiency virus type 1 (HIV-1) and the simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagmTAN), unlike human Apobec3DE, which has poor antiviral activity against these same viruses. This difference between human and chimpanzee Apobec3DE in the ability to restrict retroviruses is not due to different levels of Apobec3DE protein incorporation into virions but rather to the ability of Apobec3DE to deaminate the viral genome in target cells. We further show that Apobec3DE rapidly evolved in chimpanzee ancestors approximately 2 to 6 million years ago and that this evolution drove the increased breadth of chimpanzee Apobec3DE antiviral activity to its current high activity against some lentiviruses. Despite a difference in target specificities between human and chimpanzee Apobec3DE, Apobec3DE is likely to currently play a role in host defense against retroelements in both species.

Differential virus restriction patterns of rhesus macaque and human APOBEC3A: implications for lentivirus evolution.

The human apolipoprotein B mRNA editing enzyme catalytic peptide-like 3 (APOBEC3; A3) family of proteins (A3A-H) are known to restrict various retroviruses and retroelements, but the full complement of rhesus macaque A3 proteins remains unclear. We report the isolation and characterization of the hA3A homologue from rhesus macaques (rhA3A) and show that the rhesus macaque and human A3 genes are orthologous. RhA3A is expressed at high levels in activated CD4+ T cells, is widely expressed in macaque tissues, and is degraded in the presence of the human immunodeficiency virus (HIV-1) and simian-human immunodeficiency virus (SHIV) genomes. Our results indicate that rhA3A is a potent inhibitor of SHIVΔvif and to a lesser extent HIV-1Δvif. Unlike hA3A, rhA3A did not inhibit adeno-associated virus 2 (AAV-2) replication and L1 retrotransposition. These data suggest an evolutionary switch in primate A3A virus specificity and provide the first evidence that a primate A3A can inhibit lentivirus replication.

Intron-containing type I and type III IFN coexist in amphibians: refuting the concept that a retroposition event gave rise to type I IFNs.

Type I and III IFNs are structurally related cytokines with similar antiviral functions. They have different genomic organizations and bind to distinct receptor complexes. It has been vigorously debated whether the recently identified intron containing IFN genes in fish and amphibians belong to the type I or III IFN family or diverged from a common ancestral gene, that subsequently gave rise to both types. In this report, we have identified intron containing type III IFN genes that are tandemly linked in the Xenopus tropicalis genome and hence demonstrate for the first time that intron containing type I and III genes diverged relatively early in vertebrate evolution, and at least by the appearance of early tetrapods, a transition period when vertebrates migrated from an aquatic environment to land. Our data also suggest that the intronless type I IFN genes seen in reptiles, birds, and mammals have originated from a type I IFN transcript via a retroposition event that led to the disappearance of intron-containing type I IFN genes in modern vertebrates. In vivo and in vitro studies in this paper show that the Xenopus type III IFNs and their cognate receptor are ubiquitously expressed in tissues and primary splenocytes and can be upregulated by stimulation with synthetic double-stranded RNA, suggesting they are involved in antiviral defense in amphibians.

Epigenetics lessons from twins: prospects for autoimmune disease.

The existence of phenotypic differences between monozygotic (MZ) twins is a prime case where the relationship between genetic determinants and environmental factors is illustrated. Although virtually identical from a genetic point of view, MZ twins show a variable degree of discordance with respect to different features including susceptibility to disease. Discordance has frequently been interpreted in terms of the impact of the environment with genetics. In this sense, accumulated evidence supports the notion that environmental factors can have a long-term effect on epigenetic profiles and influence the susceptibility to disease. In relation with autoimmune diseases, the identification of DNA methylation changes in individuals who develop the disease, and the influence of inhibitors of DNA methyltransferases and histone modification enzymes in the development of autoimmunity are attracting the attention of researchers in the epigenetics field. In this context, the study of discordant MZ twins constitutes an attractive model to further investigate the epigenetic mechanisms involved in their development as well as to dissect the contribution of environmental traits. The implications of novel strategies to map epigenetic profiles and how the use of MZ twins can contribute to dissect the epigenetic component of autoimmune disease are discussed.

APOBEC3G: an intracellular centurion.

The intrinsic antiretroviral factor APOBEC3G (A3G) is highly active against HIV-1 and other retroviruses. In different cell types, A3G is expressed in high-molecular-mass (HMM) RNA- protein complexes or low-molecular-mass (LMM) forms displaying different biological activities. In resting CD4 T cells, a LMM form of A3G potently restricts HIV-1 infection soon after virion entry. However, when T cells are activated, LMM A3G is recruited into HMM complexes that include Staufen-containing RNA granules. These complexes are probably nucleated by the induced expression of Alu/hY retroelement RNAs that accompany T-cell activation. HMM A3G sequesters these retroelement RNAs away from the nuclear long interspersed nuclear element-derived enzymes required for Alu/hY retrotransposition. Human immunodeficiency virus (HIV) exploits this 'window of opportunity' provided by the loss of LMM A3G in activated CD4 T cells to productively infect these cells. During HIV virion formation, newly synthesized LMM A3G is preferentially encapsidated but only under conditions where Vif is absent and thus not able to target A3G for proteasome-mediated degradation. Together, these findings highlight the discrete functions of the different forms of A3G. LMM A3G opposes the external threat posed by exogenous retroviruses, while HMM A3G complexes oppose the internal threat posed by the retrotransposition of select types of retroelements.

Linking retroelements to autoimmunity.

In this issue, Stetson et al. (2008) report a mechanism by which host cells avert an autoimmune response to self-nucleic acids. They show that the nuclease Trex1 prevents the accumulation of DNA derived from endogenous retroelements that, if left unchecked, trigger elevated production of type I interferons leading to autoimmunity.

Trex1 prevents cell-intrinsic initiation of autoimmunity.

Detection of nucleic acids and induction of type I interferons (IFNs) are principal elements of antiviral defense but can cause autoimmunity if misregulated. Cytosolic DNA detection activates a potent, cell-intrinsic antiviral response through a poorly defined pathway. In a screen for proteins relevant to this IFN-stimulatory DNA (ISD) response, we identify 3' repair exonuclease 1 (Trex1). Mutations in the human trex1 gene cause Aicardi-Goutieres syndrome (AGS) and chilblain lupus, but the molecular basis of these diseases is unknown. We define Trex1 as an essential negative regulator of the ISD response and delineate the genetic pathway linking Trex1 deficiency to lethal autoimmunity. We show that single-stranded DNA derived from endogenous retroelements accumulates in Trex1-deficient cells, and that Trex1 can metabolize reverse-transcribed DNA. These findings reveal a cell-intrinsic mechanism for initiation of autoimmunity, implicate the ISD pathway as the cause of AGS, and suggest an unanticipated contribution of endogenous retroelements to autoimmunity.