Exogenous Retroviruses Research Articles

Abstract IA02: Functional role of endogenous retroviruses in cancer immunity

Abstract Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections of germ cells, which integrated into the genome of mammals and are inherited in a Mendelian manner. ERVs belong to the class of retro-elements possessing three main viral genes: group-specific antigen (gag), polymerase (pol) and envelope (env) flanked by two long terminal repeats (LTR). More than 8% of the human genome is considered of retroviral origin. Most ERV genes are protein non-functional due to recombination, mutations and deletions; however some ERV gag, pol and env genes produce functional proteins. Although largely gene defective, the LTRs of ERVs retained their regulatory activities resulting in high transcriptional levels; and in addition represent most of the ~10,000 annotated long non-coding (lnc) RNAs. In somatic cells ERVs are generally silent; however we and others demonstrated ERVs become activated during tumorigenesis. Recently we showed that induction of immune responses in human cancer cells by DNA methyltransferase inhibitors (DNMTi), like 5-azacytidine (Aza) and 2'-deoxy-5-azacytidine (Dac) is primarily due to the activation of (hyper)-methylated ERVs (Chiappinelli et al. 2015, Cell 162: 974-86). Furthermore, the unique genomic architecture of ERVs with 5 and 3 LTRs induced sense and antisense RNAs leading to formation of cytosolic dsRNA, which activated the cellular viral RNA sensing and defense pathway (like TLR3 and MAVS) triggering the type I interferon (IFN) response. This kind of response following ERV activation is called viral mimicry or endogenous viremia. Using ovarian carcinoma (OVCA) cell lines as a model, we showed that Aza significantly induced multiple ERV families and IFN stimulated genes (ISG). The induction was due to the formation of dsRNA, which was abolished with RNAse III (hydrolyzing dsRNA) and TLR3 or MAVS specific inhibition. ISGs were also directly induced by transfected ERV genes. Subsequently, a cohort of primary human serous OVCA tissues was analyzed and a significant positive correlation between ERV and ISG expression could be demonstrated. We and others previously showed that treatment with the DNMTi Aza positively contributed to immune checkpoint blockade therapy in a mouse model and in human cancers. Like exogenous retroviruses, which upregulate immune checkpoint genes (ICG), we also evaluated for a possible connection between ERV induction and ICG expression in primary tumors. Presently, we demonstrate that human serous OVCA tissues express significantly higher ICGs than control ovarian tissues, with VTCN-1 (B7-H4) as the highest expressed ICG. Using serous OVCA cell lines transfected with different ERV genes we could show an early induction of ICGs, primarily LGALS9 and PD-L1. Further analysis of tumor infiltrating immune cells using the same serous OVCA cohort and multiplex immunohistochemistry (IHC), we detect in some tumors high levels of tumor-associated CD79a, CD3+/CD8- and CD3+/CD8+ cells. Moreover, we find a direct link between ISG, ICG and infiltrating immune cells in OVCA. Analyzing other human tumors, e.g. brain tumors, like glioblastoma multiforme (GBM) and giant cell GBM (gcGBM) also demonstrate a significant correlation between high ERV induction and ISG as well as ICG expression. In contrast to serous OVCA, PD1 and VTCN-1 were the highest expressed ICG in GBM and gcGBM, which could also be shown with ERV transfected GBM cell lines. Using IHC gcGBM shows a high level of immune cell infiltration, which associated with giant cells and ICG expression, like PD-L1. Interestingly, the highest ERV expression was found in gcGBM, which correlated with endomitosis and cell fusion leading to giant nuclei and multinucleated cells as also shown with a stable ERV transfected GBM cell line. In summary, DNMTi mediated loss of methylation induced ERVs forming dsRNA in tumor cells and stimulated the innate immune system via ISGs. ERV RNA per se induced ISGs, but also ICGs similar to exogenous retroviruses in order to escape the immune system. Different upregulated ERVs, ISGs and ICGs were found in different cancers, like serous OVCA, GBM and gcGBM indicating diverse tumor and tumor microenvironment signatures. Thus ERVs have an ambivalent role in tumorigenesis, where tumor microenvironmental factors tip the balance being detrimental by generating immune evasion or beneficial for cancer therapy by stimulating the innate immune system. Citation Format: Reiner Strick. Functional role of endogenous retroviruses in cancer immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA02.

Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation.

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells. Previous work identified one of the youngest feline ERV groups, ERV-DC, and reported that two ERV-DC loci, ERV-DC10 and ERV-DC18 (ERV-DC10/DC18), can replicate in cultured cells. Here, we identified another replication-competent provirus, ERV-DC14, on chromosome C1q32. ERV-DC14 differs from ERV-DC10/DC18 in its phylogeny, receptor usage, and, most notably, transcriptional activities; although ERV-DC14 can replicate in cultured cells, it cannot establish a persistent infection owing to its low transcriptional activity. Furthermore, we examined ERV-DC transcription and its regulation in feline tissues. Quantitative reverse transcription-PCR (RT-PCR) detected extremely low ERV-DC10 expression levels in feline tissues, and bisulfite sequencing showed that 5' long terminal repeats (LTRs) of ERV-DC10/DC18 are significantly hypermethylated in feline blood cells. Reporter assays found that the 5'-LTR promoter activities of ERV-DC10/DC18 are high, whereas that of ERV-DC14 is low. This difference in promoter activity is due to a single substitution from A to T in the LTR, and reverse mutation at this nucleotide in ERV-DC14 enhanced its replication and enabled it to persistently infect cultured cells. Therefore, ERV-DC LTRs can be divided into two types based on this nucleotide, the A type or T type, which have strong or attenuated promoter activity, respectively. Notably, ERV-DCs with T-type LTRs, such as ERV-DC14, have expanded in the cat genome significantly more than A-type ERV-DCs, despite their low promoter activities. Our results provide insights into how the host controls potentially infectious ERVs and, conversely, how ERVs adapt to and invade the host genome. The domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity, such as ERV-DC10 and ERV-DC18, were suppressed by promoter methylation in LTRs. On the other hand, replication-competent viruses with weak promoter activity, such as ERV-DC14, seemed to escape strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity, such as ERV-DC14, have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve the understanding of the host-virus conflict and how ERVs adapt in their hosts over time.

High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has “captured” a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated.

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Classification and characterization of human endogenous retroviruses; mosaic forms are common.

BackgroundHuman endogenous retroviruses (HERVs) represent the inheritance of ancient germ-line cell infections by exogenous retroviruses and the subsequent transmission of the integrated proviruses to the descendants. ERVs have the same internal structure as exogenous retroviruses. While no replication-competent HERVs have been recognized, some retain up to three of four intact ORFs. HERVs have been classified before, with varying scope and depth, notably in the RepBase/RepeatMasker system. However, existing classifications are bewildering. There is a need for a systematic, unifying and simple classification. We strived for a classification which is traceable to previous classifications and which encompasses HERV variation within a limited number of clades.ResultsThe human genome assembly GRCh 37/hg19 was analyzed with RetroTector, which primarily detects relatively complete Class I and II proviruses. A total of 3173 HERV sequences were identified. The structure of and relations between these proviruses was resolved through a multi-step classification procedure that involved a novel type of similarity image analysis (“Simage”) which allowed discrimination of heterogeneous (noncanonical) from homogeneous (canonical) HERVs. Of the 3173 HERVs, 1214 were canonical and segregated into 39 canonical clades (groups), belonging to class I (Gamma- and Epsilon-like), II (Beta-like) and III (Spuma-like). The groups were chosen based on (1) sequence (nucleotide and Pol amino acid), similarity, (2) degree of fit to previously published clades, often from RepBase, and (3) taxonomic markers. The groups fell into 11 supergroups. The 1959 noncanonical HERVs contained 31 additional, less well-defined groups. Simage analysis revealed several types of mosaicism, notably recombination and secondary integration. By comparing flanking sequences, LTRs and completeness of gene structure, we deduced that some noncanonical HERVs proliferated after the recombination event. Groups were further divided into envelope subgroups (altogether 94) based on sequence similarity and characteristic “immunosuppressive domain” motifs. Intra and inter(super)group, as well as intraclass, recombination involving envelope genes (“env snatching”) was a common event. LTR divergence indicated that HERV-K(HML2) and HERVFC had the most recent integrations, HERVL and HUERSP3 the oldest.ConclusionsA comprehensive HERV classification and characterization approach was undertaken. It should be applicable for classification of all ERVs. Recombination was common among HERV ancestors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0232-y) contains supplementary material, which is available to authorized users.

Human Endogenous Retroviruses as Pathogenic Factors in the Development of Schizophrenia.

Schizophrenia is a complex disorder, characterized by the interplay between genetic and environmental factors. Human endogenous retroviruses (HERVs), genetic elements that originated from infections by exogenous retroviruses millions of years ago, comprise ~8% of the human genome. Here, we provide a comprehensive review of accumulating evidence, detailing HERV aberrancies associated with schizophrenia. Studies examining the genome, transcriptome, and proteome of individuals with schizophrenia provide data that support the association of these viral elements with the disorder. Molecular differences can be found within the central nervous system and peripheral tissues. However, additional studies are needed to substantiate the reported link and to address several discrepancies among previous investigations. We further discuss potentially relevant pathogenic mechanisms to the development of schizophrenia.

The Role of Human Endogenous Retroviruses (HERV-K) in the Pathogenesis of Human Cancers

Transmission of oncogenic retroviruses demonstrated in 1908. Much work was done around that period since by other researchers who demonstrated transfer of Sarcoma in chickens through filtrates. Although the name ‘retroviruses’ was coined as late as 1974, retroviral diseases were distinguished much earlier. Human endogenous retroviruses (HERVs) are natural components of the human genome and are considered remnants of ancient germ line infections by exogenous retroviruses. The human genome sequencing project revealed that 8 to 9% of the human genome is of retroviral origin. About 800 of these elements (class II HERVs) are beta-retrovirus-like and therefore distantly related to exogenous MMTV (an accepted aetiological agent for mammary tumour in mice). The majority of HERVs are noninfectious and replication-defective retroviral fossils. Some members of each HERV family however have been found to still be transcriptionally active. Furthermore, tissue-specific HERV expression profiles could be established for all human tissues investigated so far, confirming that HERVs are permanent components of the human transcriptome. A prevalence of HERV transcripts, in particular class II elements, such as members of the HML-2 family have been reported for various cancer tissues. In this review we focus our attention therefore on the association of class II HERVs with cancers. We also discuss the importance of this group of HERVs to humans, their relationship with other triggering factors (e.g. other viruses); and if there is to date a definitive causal role in cancer. We outlined the future perspectives with respect to HERVs and its contribution to human cancers, the methods of diagnosing and prognosticating; and plans to forestall attendant disease linked to these viruses by vaccine route, for example. The active sub-species purported to cause diseases in humans are the HERV-K and HERV-W.

Multiple Sclerosis between Genetics and Infections: Human Endogenous Retroviruses in Monocytes and Macrophages.

The etiology of multiple sclerosis (MS) is still unknown, but there is strong evidence that genetic predisposition associated with environmental factors can trigger the disease. An estimated 30 million years ago, exogenous retroviruses are thought to have integrated themselves into human germ line cells, becoming part of human DNA and being transmitted over generations. Usually such human endogenous retroviruses (HERVs) are silenced or expressed at low levels, but in some pathological conditions, such as MS, their expression is higher than that in the healthy population. Three HERV families have been associated with MS: HERV-H, HERV-K, and HERV-W. The envelope protein of MS-associated retrovirus (MSRV) from the HERV-W family currently has the strongest evidence as a potential trigger for MS. In addition to expression in peripheral immune cells, MSRV is expressed in monocytes and microglia in central nervous system lesions of people with MS and, through the activation of toll-like receptor 4, it has been shown to drive the production of proinflammatory cytokines, reduction of myelin protein expression, and death of oligodendrocyte precursors. In conclusion, the association between HERVs and MS is well documented and a pathological role for MSRV in MS is plausible. Further studies are required to determine whether the presence of these HERVs is a cause or an effect of immune dysregulation in MS.

The potential role of retroviruses in autoimmunity.

In the last 20 years research in Immunology underwent fundamental changes. Most importantly, the identification of the key role of innate immune pattern recognition receptors (PRRs) that recognize evolutionarily conserved molecular patterns on infectious pathogens. This results in priming of innate immune cells, which in turn activate and direct the adaptive immune response. Progress in innate immune recognition instigated the current working hypothesis, that recognition of endogenous ligands by PRRs results in innate immune cell activation (autoinflammation) or activation of adaptive cells, with self-reactive antigen receptors (autoimmunity). In particular, nucleic acid-sensing innate immune receptors seem to be prime candidates for a mechanistic understanding of autoreactive activation of the immune system. However, it remains uncertain what the actual source of nucleic acid ligands is and what other signals are needed to drive activation of autoreactive innate immune cells and break self-tolerance of the adaptive immune system. Here, I will review our present understanding about whether the infection with exogenous retroviruses or the reactivation of endogenous retroviruses might play an etiological role in certain autoimmune conditions of humans and murine experimental models.

Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Cooption and Domestication.

Endogenous retroviruses (ERVs) are remnants of ancestral retroviral infections of germ cells. Retroviral endogenization is an adaptation process for the host genome, and ERVs are gradually attenuated or inactivated by mutation. However, some ERVs that have been "domesticated" by their hosts eventually gain physiological functions, such as placentation or viral resistance. We previously reported the discovery of Refrex-1, a soluble antiretroviral factor in domestic cats that specifically inhibits infection by feline leukemia virus subgroup D (FeLV-D), a chimeric virus of FeLV, and a feline ERV, ERV-DC. Refrex-1 is a truncated envelope protein (Env) encoded by both ERV-DC7 and ERV-DC16 proviral loci. Here, we reconstituted ancestral and functional Env from ERV-DC7 and ERV-DC16 envelope genes (env) by inducing reverse mutations. Unexpectedly, ERV-DC7 and ERV-DC16 full-length Env (ERV-DC7 fl and ERV-DC16 fl), reconstructed by removing stop codons, did not produce infectious viral particles. ERV-DC7 fl and ERV-DC16 fl were highly expressed in cells but were not cleaved into surface subunits (SU) and transmembrane subunits, nor were they incorporated into virions. G407R/N427I-A429T and Y431D substitutions within the SU C-terminal domain of ERV-DC7 fl and ERV-DC16 fl, respectively, caused these dysfunctions. The residues glycine 407 and tyrosine 431 are relatively conserved among infectious gammaretroviruses, and their substitution causes the same dysfunctions as the tested retroviruses. Our results reveal that specific mutations within the SU C-terminal domain suppressed Env cleavage and incorporation into virions and indicate that these mutations contributed to the domestication of Refrex-1 through multistep events that occurred in the postintegration period. Domestic cats are colonized with various exogenous retroviruses (exRVs), such as feline leukemia virus (FeLV), and their genomes contain numerous ERVs, some of which are replication-competent proviruses. The feline hosts, exRVs, and ERVs have complicated genetic interactions and provide an interesting field model for triangular relationships: recombination between FeLV and ERV-DC, which is a feline ERV, generated FeLV-D, a chimeric virus, and FeLV-D is restricted by Refrex-1, an antiretroviral factor corresponding to truncated Env of ERV-DC7 and ERV-DC16. Here, we reconstructed ancestral, functional Env from ERV-DC7 and ERV-DC16 env by inducing reverse mutations to elucidate how Refrex-1 was generated from its ancestor. Our results reveal that they were repeatedly inactivated by mutations preventing Env maturation. Our results provide insights into how ERVs were "domesticated" by their hosts and identify the mutations that mediated these evolutions. Notably, experiments that restore inactivated ERVs might uncover previously unrecognized features or properties of retroviruses.

Koala Retroviruses: Evolution and Disease Dynamics.

A retroviral etiology for malignant neoplasias in koalas has long been suspected. Evidence for retroviral involvement was bolstered in 2000 by the isolation of a koala retrovirus (KoRV), now termed KoRV-A. KoRV-A is an endogenous retrovirus-a retrovirus that infects germ cells-a feature that makes it a permanent resident of the koala genome. KoRV-A lacks the genetic diversity of an exogenous retrovirus, a quality associated with the ability of a retrovirus to cause neoplasias. In 2013, a second KoRV isolate, KoRV-B, was obtained from koalas with lymphomas in the Los Angeles Zoo. Unlike KoRV-A, which is present in the genomes of all koalas in the United States, KoRV-B is restricted in its distribution and is associated with host pathology (neoplastic disease). Here, our current understanding of the evolution of endogenous and exogenous KoRVs, and the relationship between them, is reviewed to build a perspective on the future impact of these viruses on koala sustainability.

Wide distribution and ancient evolutionary history of simian foamy viruses in New World primates.

BackgroundAlthough simian foamy viruses (SFV) are the only exogenous retroviruses to infect New World monkeys (NWMs), little is known about their evolutionary history and epidemiology. Previous reports show distinct SFVs among NWMs but were limited to small numbers of captive or wild monkeys from five (Cebus, Saimiri, Ateles, Alouatta, and Callithrix) of the 15 NWM genera. Other studies also used only PCR testing or serological assays with limited validation and may have missed infection in some species. We developed and validated new serological and PCR assays to determine the prevalence of SFV in blood specimens from a large number of captive NWMs in the US (n = 274) and in captive and wild-caught NWMs (n = 236) in Peruvian zoos, rescue centers, and illegal trade markets. Phylogenetic and co-speciation reconciliation analyses of new SFV polymerase (pol) and host mitochondrial cytochrome B sequences, were performed to infer SFV and host co-evolutionary histories.Results124/274 (45.2 %) of NWMs captive in the US and 59/157 (37.5 %) of captive and wild-caught NWMs in Peru were SFV WB-positive representing 11 different genera (Alouatta, Aotus, Ateles, Cacajao, Callithrix, Cebus, Lagothrix, Leontopithecus, Pithecia, Saguinus and Saimiri). Seroprevalences were lower at rescue centers (10/53, 18.9 %) compared to zoos (46/97, 47.4 %) and illegal trade markets (3/7, 8/19, 42.9 %) in Peru. Analyses showed that the trees of NWM hosts and SFVs have remarkably similar topologies at the level of species and sub-populations suggestive of co-speciation. Phylogenetic reconciliation confirmed 12 co-speciation events (p < 0.002) which was further supported by obtaining highly similar divergence dates for SFV and host genera and correlated SFV-host branch times. However, four ancient cross-genus transmission events were also inferred for Pitheciinae to Atelidae, Cacajao to ancestral Callithrix or Cebus monkeys, between Callithrix and Cebus monkeys, and Lagothrix to Alouatta.ConclusionsWe demonstrate a broad distribution and stable co-speciation history of SFV in NWMs at the species level. Additional studies are necessary to further explore the epidemiology and natural history of SFV infection of NWMs and to determine the zoonotic potential for persons exposed to infected monkeys in captivity and in the wild.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0214-0) contains supplementary material, which is available to authorized users.

Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads.

From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses.

Endogenous Retroviruses in the Genomics Era.

Endogenous retroviruses comprise millions of discrete genetic loci distributed within the genomes of extant vertebrates. These sequences, which are clearly related to exogenous retroviruses, represent retroviral infections of the deep past, and their abundance suggests that retroviruses were a near-constant presence throughout the evolutionary history of modern vertebrates. Endogenous retroviruses contribute in myriad ways to the evolution of host genomes, as mutagens and as sources of genetic novelty (both coding and regulatory) to be acted upon by the twin engines of random genetic drift and natural selection. Importantly, the richness and complexity of endogenous retrovirus data can be used to understand how viruses spread and adapt on evolutionary timescales by combining population genetics and evolutionary theory with a detailed understanding of retrovirus biology (gleaned from the study of extant retroviruses). In addition to revealing the impact of viruses on organismal evolution, such studies can help us better understand, by looking back in time, how life-history traits, as well as ecological and geological events, influence the movement of viruses within and between populations.

Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms.

BackgroundKoala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag.ResultsIn this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results.ConclusionsThese results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0193-1) contains supplementary material, which is available to authorized users.

Abstract 2966A: A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues

Abstract Human endogenous retrovirus (HERV) is a member of LTR-retrotransposon family. Retroviral invasions are thought to have occurred in the germ cell lines of human or human ancestors, and the fixed retroviral elements are recognized as HERV. A complete HERV sequence contains two long terminal repeats (LTRs) located at both ends. Because intact LTRs possess the highly conserved transcriptional promoter- and enhancer-sequences, to investigate insertional variations of HERV is a key process for understanding the etiology of human genetic diseases that remain unclear with the analyses targeting single nucleotide variations. However, genome-wide investigation of repetitive sequences including HERV is methodologically difficult, and comprehensive understanding of insertional variations of HERV, including a somatic retrotransposition of HERV, remains poor. In the previous study, we have identified two novel insertional polymorphisms of solo LTRs using the inverse PCR and cloning methods, and one of them was involved with the susceptibility to lung adenocarcinoma. Here, we detected multiple novel LTR loci in the human cancer tissues by developing the high-throughput sequencing method combined with inverse PCR. In this system, to preferentially detect insertional variations, primer sequences were designed based on the conserved region of the LTRs which have an identical sequence to the mate within the same HML-2 proviruse, because almost of known HERV insertional polymorphisms are found in HML-2, which is thought to be the most recently endogenized element of HERV, and the presence of an identical LTR sequence at both ends is necessary for the retrotransposition of exogenous retrovirus. The sequence reads generated from the high-throughput sequencer were trimmed, and the fragments consistent with the inverse PCR process were selected. Following the mapping to the reference genome, the sites not corresponding to known LTR loci were sorted out and subjected to the filtration to exclude the artificial fragments. As the result, some candidates of novel LTR loci were detected, and several of them were verified to be actually present both in tumor and non-tumor genomes using the Sanger method, suggesting they are insertional polymorphisms. By using this detection system, we are now challenging to investigate a novel LTR site specifically detected in tumor genome. Citation Format: Tomoaki Kahyo, Hidetaka Yamada, Hong Tao, Yusuke Inoue, Nobuya Kurabe, Haruhiko Sugimura. A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2966A. doi:10.1158/1538-7445.AM2015-2966A

Endogenization of mouse mammary tumor virus (MMTV)-like elements in genomes of pikas (Ochotona sp.)

Despite the finding in European rabbit and other leporid genomes of the first ever described endogenous lentivirus and of a European rabbit exclusive endogenous gammaretrovirus, until now no exogenous retroviruses have been isolated in Lagomorpha species. Nevertheless, looking for the presence of endogenous retroviruses (ERVs) in the species genomes could lead to the discovery of retroviral lineages yet to be found in Lagomorpha.Different mammalian genomes harbor endogenous viral sequences phylogenetically close to the betaretrovirus mouse mammary tumor virus (MMTV), propelling us to look for such retroviral “fossil” in American pika (Ochotona princeps) and European rabbit (Oryctolagus cuniculus) genomes. By performing genomic mining using MMTV gag and LTR as query sequences, we found that such viral elements were absent from the European rabbit genome. Oppositely, significant matches were found in American pika, and more importantly, a nearly complete MMTV-like virus (Pika-BERV) was identified.Using Pika-BERV gag and LTR as templates, we found similar sequences endogenized in different pika (Ochotona sp.) species. The orthology of the LTR flanking region between some pika species supported shared ancestry of specific endogenous betaretroviruses, while in other pika species similar sequences, but not orthologous, should have resulted from independent insertions.Our study supports the possible existence of infecting exogenous betaretroviruses for a long term, after the divergence of Ochotonidae from Leporidae, but yet to be identified.

The histone methyltransferase SETDB1 represses endogenous and exogenous retroviruses in B lymphocytes

Genome stability relies on epigenetic mechanisms that enforce repression of endogenous retroviruses (ERVs). Current evidence suggests that distinct chromatin-based mechanisms repress ERVs in cells of embryonic origin (histone methylation dominant) vs. more differentiated cells (DNA methylation dominant). However, the latter aspect of this model has not been tested. Remarkably, and in contrast to the prevailing model, we find that repressive histone methylation catalyzed by the enzyme SETDB1 is critical for suppression of specific ERV families and exogenous retroviruses in committed B-lineage cells from adult mice. The profile of ERV activation in SETDB1-deficient B cells is distinct from that observed in corresponding embryonic tissues, despite the loss of repressive chromatin modifications at all ERVs. We provide evidence that, on loss of SETDB1, ERVs are activated in a lineage-specific manner depending on the set of transcription factors available to target proviral regulatory elements. These findings have important implications for genome stability in somatic cells, as well as the interface between epigenetic repression and viral latency.

인간 내성 리트로 바이러스(HERV)와 인간 면역 결핍 바이러스(HIV)의 상관관계

Retroviruses genes have been inserted into the human genome for millions of years. These retroviruses are now inactive due to mutations such as deletions or nonsense mutations. After mutation, retroviruses eventually became fixed in the genome in their endogenous forms and existed as traces of ancient viruses. These retroviruses are called endogenous retroviruses (ERVs), with the human form known as human endogenous retrovirus. HERV cannot become a fully active virus, but a number of viral proteins or even virus particles are expressed under various conditions. Compared to endogenous retroviruses, some exogenous retroviruses are still infectious and can threaten human life. Among these, human immunodeficiency virus (HIV) is one of the most well-known and best-studied. Recent studies have shown some elements of HERV were activated by HIV infection and interact with HIV-derived proteins. In addition, many studies have attempted to use HERV as vaccination against HIV infection. This review will describe the regulation and interaction between HERV and HIV infection and mention the development of vaccines and therapeutic agents against HIV infection by using HERV elements.

Human endogenous retroviruses sustain complex and cooperative regulation of gene-containing loci and unannotated megabase-sized regions.

BackgroundEvidence suggests that some human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively ERVs) regulate the expression of neighboring genes in normal and disease states; e.g. the human globin locus is regulated by an ERV9 that coordinates long-range gene switching during hematopoiesis and activates also intergenic transcripts. While complex transcription regulation is associated with integration of certain exogenous retroviruses, comparable regulation sustained by ERVs is less understood.FindingsWe analyzed ERV transcription using ERV9 consensus sequences and publically available RNA-sequencing, chromatin immunoprecipitation with sequencing (ChIP-seq) and cap analysis gene expression (CAGE) data from ENCODE. We discovered previously undescribed and advanced transcription regulation mechanisms in several human reference cell lines. We show that regulation by ERVs involves long-ranging activations including complex RNA splicing patterns, and transcription of large unannotated regions ranging in size from several hundred kb to around 1 Mb. Moreover, regulation was found to be cooperatively sustained in some loci by multiple ERVs and also non-LTR repeats.ConclusionOur analyses show that endogenous retroviruses sustain advanced transcription regulation in human cell lines, which shows similarities to complex insertional mutagenesis effects exerted by exogenous retroviruses. By exposing previously undescribed regulation effects, this study should prove useful for understanding fundamental transcription mechanisms resulting from evolutionary acquisition of retroviral sequence in the human genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0161-9) contains supplementary material, which is available to authorized users.