Exogenous Retroviruses Research Articles

Expression of endogenous beta retroviruses and Hyal-2 mRNA in immune organs of fetuses and lambs

Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

Natural Infection and Transmission of a Retrovirus Closely Related to Myeloblastosis-Associated Virus Type 1 in Egg-Type Chickens

Myeloblastosis-associated virus type 1 (MAV-1) is an exogenous avian retrovirus with oncogenic potential. MAV-1 was detected in young chicks hatching from eggs produced by an experimental genetic line of egg-type chickens. Transmissibility of MAV-1 had not been documented previously. This investigation was intended to partially characterize the virus involved and to study its transmissibility and oncogenicity in naturally and contact-infected chickens. Commercially produced white and brown layer pullets free of exogenous avian leukosis viruses were commingled at hatch with naturally MAV-1-infected chickens. The original MAV-1-infected chickens were discarded after approximately 8 wk, and the contact-exposed chickens were maintained in isolation for 36 wk. Young specific-pathogen-free (SPF) single comb white leghorn chickens were added to the group to study possible horizontal transmission of MAV-1 in young chickens. Upon weekly virus isolation attempts, MAV-1 was readily isolated from the contact-exposed white layers but not from the brown layers between 36 and 53 wk of age (18 wk in total). Three-week-old SPF chickens were readily infected with MAV-1 by contact as early as 1 wk postexposure. Throughout 22 hatches derived from the white and brown MAV-1-contact-exposed layers (between 36 and 53 wk of age), MAV-1 was frequently detected in the white layer progeny, whereas the virus was seldom isolated from the progeny produced by the brown layers during the same 18-wk period. MAV-1 induced a persistent infection in some of the SPF chickens that were exposed by contact at 3 wk of age. Gross tumors were not detected in any of the originally infected experimental chickens at 8 wk of age, in the contact-exposed brown or white layers at the termination of the study at 53 wks of age, or in the contact-exposed SPF chickens at the end of the study at 12 wk of age. Exogenous avian leukosis-related viruses may still be detected in egg-type chickens, emphasizing the importance of thorough screening before incorporation of experimental genetic material into commercial genetic lines of egg-type chickens.

Epidemiological survey of a captive Chinese rhesus macaque breeding colony in Yunnan for SRV, STLV and BV

Nonhuman primates are critical resources for biomedical research. Rhesus macaque is a popularly used laboratory nonhuman primate that share many characteristics with humans. However, rhesus macaques are the natural host of two exogenous retroviruses, SRV (simian type D retrovirus) and STLV (simian T lymphotropic virus). SRV and STLV may introduce potentially significant confounding factors into the study of AIDS model. Moreover, B virus (ceropithecine herpesvirus 1) is likely to harm not only rhesus macaque but also humans in experiments involving rhesus macaque. Yunnan province has large-scale breeding colonies of Chinese rhesus macaque. Therefore there is an urgent need for SPF Chinese rhesus macaque colonies. Here we investigated SRV, STLV and BV infections in 411 Chinese rhesus macaque by PCR technique. The results showed that the prevalence of SRV, STLV and BV among Chinese rhesus macaque breeding colony was 19.71% (81/411), 13.38% (55/411) and 23.11% (95/411), respectively. Comparison of viruses infection in different age-groups and male/female of Chinese rhesus macaque was also analyzed. This study will contribute to establishment of SPF Chinese rhesus macaque breeding colony.

Abstract A62: Human endogenous retrovirus activation in prostate cancer: Association with disease progression

Abstract Introduction: In recent years scientists have observed that several tumor types, including prostate cancer, show increased expression of human endogenous retrovirus (HERV) when compared to normal tissue. The HERVs originated from germ cell infections by exogenous retroviruses during the course of evolution and became incorporated into the human genome. These elements are widely dispersed throughout the genome and are estimated to comprise of greater than >8% of genomic material. The HERV-K family, evolutionarily the youngest HERV, is the only HERV family with complete open reading frames for all viral genes, and thus are the most likely to be biologically active and potentially pathogenic. The HERV-K provirus encodes for the gag, pol and env genes flanked by two long terminal repeat regions. Both HERV-K type I and HERV-K type II proviruses exist: Type II proviruses are complete (and a such are chronological “older” with regard to their insertion into the genome), while Type I proviruses contain a 292-bp deletion at the boundary of the pol and env genes. Hypothesis: We hypothesize that HERV-K expression is activated with advancing prostate cancer progression and that as a result of this, HERV-K leads to a sustained inflammatory response and subsequent promotion of tumor progression. We believe that HERV-K is suitable target of therapeutic intervention. Experimental Procedures: RNA sequences for type II HERV-K108 (AF074086) were blasted against type I HERV-K102 (AF164610). Sequences were 99% homologous between nucleotides 1-6492 (K108) & 1-1691 (K102) (5′LTR-gag-pro-pol region), the type 1 del in K102 occurred between nucleotides 6493-6793 of K108, subsequent nucleotides 6794-9472 (K108) & 6501-9178 (K102) were 99% homologous (env-3′LTR region). For the detection of HERV-K gag, pol and env mRNA proteins were designed in Primer3, and verified for specificity in Primer-BLAST. Levels of HERV-K gag, pol and env type I or II mRNA were quantified in RWPE1 normal immortalized prostate cells, and CRW22 androgen dependent prostate cancer cell lines, and 22Rv1, PC-3 and DU145 androgen independent prostate cancer cell lines (22Rv1 AI variant of CRW22, PC-3 and DU145 derived from prostate cancer metastasis). HERV-K gag and env protein expression was detected in cell lines by western blotting and in prostate cancer tissue microarrays of hyperplasia and adenocarcinoma using anti-HERV-K gag monoclonal antibody (MAb) and anti-HERV-K env MAb. Effects of HERV-K env inhibition in prostate cancer cells were examined by inhibiting HERV-K env using a HERV-K env targeted MAb, and assessing its effect on cell proliferation and cell invasion using the xCelligence Real-Time Cell Analyser. Results: We found that HERV-K is activated in CRW22, 22Rv1, PC3 and DU145 prostate cancer cell lines, but not in RWPE1 normal prostate cells. Additionally, we observed the HERV-K levels were highest in the bone metastasis derived PC3 and brain metastasis derived DU145 cell lines. We also found that HERV-K was differentially expressed between prostate hyperplasia and adenocarcinoma. Inhibition of HERV-K env resulted in inhibition of PC3 and DU145 cell proliferation and cell invasion. Conclusions: HERV-K is activated in prostate cancer and represents a potential therapeutic target for metastatic prostate cancer. Citation Format: Ronan Downey, Amy Burke, Francis J. Giles, Frank Sullivan, Feng Wang-Johanning, Blanaid Mee, Eoin Gaffney, Sharon Anneve Glynn. Human endogenous retrovirus activation in prostate cancer: Association with disease progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A62.

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: Different ways to counteract host-encoded restriction

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable.

HIV infection and HERV expression: a review

The human genome contains multiple copies of retrovirus genomes known as endogenous retroviruses (ERVs) that have entered the germ-line at some point in evolution. Several of these proviruses have retained (partial) coding capacity, so that a number of viral proteins or even virus particles are expressed under various conditions. Human ERVs (HERVs) belong to the beta-, gamma-, or spuma- retrovirus groups. Endogenous delta- and lenti- viruses are notably absent in humans, although endogenous lentivirus genomes have been found in lower primates. Exogenous retroviruses that currently form a health threat to humans intriguingly belong to those absent groups. The best studied of the two infectious human retroviruses is the lentivirus human immunodeficiency virus (HIV) which has an overwhelming influence on its host by infecting cells of the immune system. One HIV-induced change is the induction of HERV transcription, often leading to induced HERV protein expression. This review will discuss the potential HIV-HERV interactions.Several studies have suggested that HERV proteins are unlikely to complement defective HIV virions, nor is HIV able to package HERV transcripts, probably due to low levels of sequence similarity. It is unclear whether the expression of HERVs has a negative, neutral, or positive influence on HIV-AIDS disease progression. A positive effect was recently reported by the specific expression of HERVs in chronically HIV-infected patients, which results in the presentation of HERV-derived peptides to CD8+ T-cells. These cytotoxic T-cells were not tolerant to HERV peptides, as would be expected for self-antigens, and consequently lysed the HIV-infected, HERV-presenting cells. This novel mechanism could control HIV replication and result in a low plasma viral load. The possibility of developing a vaccination strategy based on these HERV peptides will be discussed.

Endogenous Retroviruses of Sheep: A Model System for Understanding Physiological Adaptation to an Evolving Ruminant Genome

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. The sheep genome contains 27 endogenous betaretroviruses (enJSRVs) related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. The enJSRVs can protect their host against JSRV infection by blocking early and late steps of the JSRV replication cycle. In the female reproductive tract, enJSRVs are specifically expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophectoderm growth and development. Collectively, available evidence in sheep and other mammals indicate that ERVs coevolved with their hosts for millions of years and were positively selected for biological roles in genome plasticity and evolution, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental development.

Spontaneous immunological targeting of endogenous retrovirus K is directed against the envelope protein in rhesus macaques

Endogenous retroviruses (ERVs), remnants of ancient retroviral infections, have invaded the germ line of every vertebrate species including both non-human primates and humans. Most ERVs are functionally crippled by extensive deletions, mutations and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs retain the potential for biological activity and can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. Although ERV gene products are implicated in the etiology of disease, their impact during host infection with exogenous retroviruses such as human and simian immunodeficiency virus (HIV/ SIV) remains unknown. Here, we identify the homologue of the biologically active human ERV-K, macaque ERV-K (macERV-K), which retainsintact open reading frames in the macaque genome. Indeed, we isolated a fully-spliced mRNA product encoding macERV-K Env, which contains all the structural features of a canonical retroviral Envelope protein. Furthermore, we identified robust CD8+ and CD4+ T cell responses directed at macERV-K Env in one SIV-infected Indian rhesus macaque. The macERV-K Envspecific CD4+ T cell response in this animal was larger than the entire 5lV-specific CD4+ T cell response and had a uniform effector memory phenotype, indicating continual low-level antigen exposure. In support of this, we also identified antibody responses targeting macERV-K in this animal. Although SIV infection did not appear to trigger ERV Env-specific T cell responses, these data suggest that Env proteins from ERVs can be active during infections with exogenous retroviruses. The interaction of ERV Env proteins expressed in HIV-infected cells may have major implications for the pathogenicity of HIV and warrants further investigation.

Intrinsic restriction activity by apoB mRNA editing enzyme APOBEC1 against the mobility of retroelements

A large portion of the mammalian genome is derived from ancient transposable elements. Retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate (retrotransposition), constitute a majority of these mobile genetic elements. Endogenous retroviruses are LTR-type retroelements accounting for around 8% of human or murine genomic DNA. Non-LTR members are present in extremely high copy numbers; with LINE-1 contributing to nearly 40% of human and murine genomes. These LINE-1 elements modify mammalian genomes not only through insertions, but also by indirect replication of nonautonomous retrotransposons such as SINEs. As expected, cellular machineries of vertebrate’ si nnate immunity have evolved to support a balance between retroelement insertions that cause deleterious gene disruptions and those that confer beneficial genetic diversity. The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including MLV and HIV-1, is now well established. The RNA editing family member apolipoprotein B (apo B)editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C-to-U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but it’s activity against endogenous retroelements remains unclear. Here we show that A1 family proteins can also reduce the mobility and infectivity potential of LINE-1 and LTR retrotransposons (or endogenous retroviruses) such as IAP and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism with inhibition at step prior to the integration, and was not affected by nuclear localization of the proteins. In contrast, A1 inhibits the replication of murine IAP and MusD through a DNA deamination-dependent mechanism. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in a wide range of mammalian species.

Expression of Gag and Pol from reconstructed HERV-Fc1, associated with multiple sclerosis

Background Though the etiology of Multiple Sclerosis (MS) is still obscure, Human Endogenous Retroviruses (HERVs) have long been suspected to be involved [1]. Functional studies have backed this theory, and recently our group provided more direct genetic evidence for association of MS with a provirus located on chromosome X, HERVFc1 [2]. An association with the retroviral restriction factor TRIM5 was also found. The HERV-Fc1 sequence contains the general retrovirus structure of LTR-elements and the three genes gag, pol and env. The env gene seems intact with an open reading frame (ORF). The gag ORF is terminated by two stop codons, compared to the common single stop in exogenous retroviruses. The pol frame is interrupted by a frameshift mutation and a premature stopcodon; a polymorphic C-repeat in the pol gene represents a second frameshift in some persons. HERV-Fc1 is only sparsely characterized, and we therefore aimed to investigate this provirus, especially in relation to its potential involvement in autoimmunity.

Endogenous APOBEC3B Restricts LINE-1 Retrotransposition in Transformed Cells and Human Embryonic Stem Cells

Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.

Quantitative analysis of the HERV pol gene in human tissues

Human endogenous retroviruses (HERVs) have been associated with cancer pathogenesis due to their close homology to exogenous tumorigenic retroviruses. The expression pattern of HERVs could provide important insights into the pathogenic state between HERVs and various cancers. Using the RetroTector10 program, we identified four full-length HERV elements (HML4, HERV-T, HERV-F, and HERV-R) that contain pol genes with reverse transcriptase sub domains. Through a quantitative real-time RT-PCR approach, we investigated expression levels of their pol genes in various human tissues including tumor and adjacent normal tissue samples. The HML4 and HERV-T pol genes were ubiquitously expressed in all human tissues, whereas the HERV-F pol gene was only expressed in the human placenta. In further analysis, we compared expression levels of the HERV pol genes in normal tissues and their corresponding tumor tissues. Interestingly, the HERV-F pol gene showed high expression in lung tumors while its expression level was very low in normal lung tissues (p-value = 0.007). In addition, the HERV-R pol gene showed a higher expression in stomach tumors than in normal tissues (p-value = 0.044). Thus, we suggest that the expression profiling of HERV-F and HERV-R pol genes could be used as a molecular marker system to detect human stomach and lung cancers, respectively.

Coevolution of retroelements and tandem zinc finger genes

Vertebrate genomes encode large and highly variable numbers of tandem C2H2 zinc finger (tandem ZF) transcription factor proteins. In mammals, most tandem ZF genes also encode a KRAB domain (KZNF proteins). Very little is known about what forces have driven the number and diversity of tandem ZF genes. Recent studies suggest that one role of KZNF proteins is to bind and repress transcription of exogenous retroviruses and their endogenous counterpart LTR retroelements. We report a striking correlation across vertebrate genomes between the number of LTR retroelements and the number of host tandem ZF genes. This correlation is specific to LTR retroelements and ZF genes and was not explained by covariation in other genomic features. We further show that recently active LTR retroelements are correlated with recent tandem ZF gene duplicates across vertebrates. On branches of the primate phylogeny, we find that the appearance of new families of endogenous retroviruses is strongly predictive of the appearance of new duplicate KZNF genes. We hypothesize that retroviral and LTR retroelement burden drives evolution of host tandem ZF genes. This hypothesis is consistent with previously described molecular evolutionary patterns in duplicate ZF genes throughout vertebrates. To further explore these patterns, we investigated 34 duplicate human KZNF gene pairs, all of which underwent an early burst of divergence in the major nucleotide contact residues of their ZF domains, followed by purifying selection in both duplicates. Our results support a host-pathogen model for tandem ZF gene evolution, in which new LTR retroelement challenges drive duplication and divergence of host tandem ZF genes.

Role of Human Endogenous Retroviral Long Terminal Repeats (LTRs) in Maintaining the Integrity of the Human Germ Line

Retroviruses integrate a reverse transcribed double stranded DNA copy of their viral genome into the chromosomal DNA of cells they infect. Occasionally, exogenous retroviruses infect germ cells and when this happens a profound shift in the virus host dynamic occurs. Retroviruses maintained as hereditable viral genetic material are referred to as endogenous retroviruses (ERVs). After millions of years of co-evolution with their hosts many human ERVs retain some degree of function and a few have even become symbionts. Thousands of copies of endogenous retrovirus long terminal repeats (LTRs) exist in the human genome. There are approximately 3000 to 4000 copies of the ERV-9 LTRs in the human genome and like other solo LTRs, ERV-9 LTRs can exhibit distinct promoter/enhancer activity in different cell lineages. It has been recently reported that a novel transcript of p63, a primordial member of the p53 family, is under the transcriptional control of an ERV-9 LTR [1]. The expression of different p63 transcript isoforms has been previously shown to have an important role in replenishing cutaneous epithelial stem cells and maintaining the fidelity of the female germ line [2]. In this recent report, a novel p63 transcript, designated GTAp63, is described as specifically expressed in healthy human testes and germ cell precursors of human testes but not in testicular cancer cells. The ability of ERV-9 regulatory regions to contribute to the maintenance of male germ line stability is yet another example of how ERVs have evolved to serve an important function in the physiology of their human hosts.

Independent evolution of intracellular transposons from exogenous IAP-related retroviruses in a broad range of mammalian species

Endogenous retroviruses (ERVs) are the descendants of occasional germ line invasions by exogenous retroviruses. Following these invasion events, some ERV lineages have proliferated by viral reinfection whilst others have become retrotransposon-like, losing the env gene which is required for infectivity of viral particles. The processes by which intracellular retrotransposons evolve from exogenous retroviruses remain poorly understood. The IAP (Intracisternal A-type retroviral Particles) family of ERVs provides an excellent model group in which to study this phenomenon. First identified in the Mouse genome, this well-defined and relatively recent group of ERVs have been studied extensively in vivo. We mined 36 mammalian genomes for all IAP-like ERVs, finding a total of 4913 loci within 17 genomes, mostly in several rodent species. Using these data, and taking in account the known phylogeny of the hosts, we analysed how the integrity of the env gene is related to the proliferation of IAP lineages within the host genomes. We found that loss of env is associated with the expansion of these lineages; for example in the three largest expansions accounting for more than 50% of the IAP-like loci, env has suffered extensive degradation. Our findings indicate that large copy number in IAP lineages is associated with them becoming retrotransposon-like, a process which could also apply to other, poorly studied, ERV groups. We are currently trying to identify other factors that influence this process and also to see how env loss affects other processes such as host switching.

The signal peptide of a recently integrated endogenous sheep betaretrovirus envelope plays a major role in eluding gag-mediated late restriction.

The exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with highly related and biologically active endogenous retroviruses (enJSRVs). The endogenous enJS56A1 locus possesses a defective Gag polyprotein which blocks the late replication steps of related exogenous and endogenous retroviruses by a mechanism known as JSRV late restriction (JLR). Conversely, enJSRV-26, which most likely integrated into the sheep genome less than 200 years ago, is able to escape JLR. In this study, we demonstrate that the ability of enJSRV-26 to escape JLR is due to a single-amino-acid substitution in the signal peptide (SP) of its envelope glycoprotein. We show that enJSRV-26 SP does not localize to the nucleolus, unlike the functional SPs of related exogenous and endogenous sheep betaretroviruses. In addition, enJSRV-26 SP function as a posttranscriptional regulator of viral gene expression is impaired. enJSRV-26 JLR escape relies on the presence of the functional enJS56A1 SP. Moreover, we show that the ratio between enJSRV-26 and enJS56A1 Gag is critical to elude JLR. Interestingly, we found that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus. These data further reinforce the notion that transdominant enJSRV proviruses have been positively selected in domestic sheep, and that the coevolution between endogenous and exogenous sheep betaretroviruses and their host is still occurring.

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing

Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a(-/-) cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a(-/-) line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.

Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C–U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.

Unique Spectrum of Activity of Prosimian TRIM5α against Exogenous and Endogenous Retroviruses

Lentiviruses, the genus of retrovirus that includes HIV-1, rarely endogenize. Some lemurs uniquely possess an endogenous lentivirus called PSIV ("prosimian immunodeficiency virus"). Thus, lemurs provide the opportunity to study the activity of host defense factors, such as TRIM5α, in the setting of germ line invasion. We characterized the activities of TRIM5α proteins from two distant lemurs against exogenous retroviruses and a chimeric PSIV. TRIM5α from gray mouse lemur, which carries PSIV in its genome, exhibited the narrowest restriction activity. One allelic variant of gray mouse lemur TRIM5α restricted only N-tropic murine leukemia virus (N-MLV), while a second variant restricted N-MLV and, uniquely, B-tropic MLV (B-MLV); both variants poorly blocked PSIV. In contrast, TRIM5α from ring-tailed lemur, which does not contain PSIV in its genome, revealed one of the broadest antiviral activities reported to date against lentiviruses, including PSIV. Investigation into the antiviral specificity of ring-tailed lemur TRIM5α demonstrated a major contribution of a 32-amino-acid expansion in variable region 2 (v2) of the B30.2/SPRY domain to the breadth of restriction. Data on lemur TRIM5α and the prediction of ancestral simian sequences hint at an evolutionary scenario where antiretroviral specificity is prominently defined by the lineage-specific expansion of the variable loops of B30.2/SPRY.

Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3

After fixation in the human genome, human endogenous retroviruses (HERVs) are bona fide cellular genes despite their exogenous origin. To be able to spread within the germ line and the early embryo, the ancient retroviral promoters must have adapted to the requirements for expression in these cell types. We describe that in contrast to the case for current exogenous retroviruses, which replicate in specific somatic cells, the long terminal repeat (LTR) of the human endogenous retrovirus HERV-K acts as a TATA- and initiator element-independent promoter with a variable transcription start site. We present evidence that the HERV-K LTR is regulated by the transcription factors Sp1 and Sp3. Mutating specific GC boxes, which are binding sites for Sp proteins, and knocking down Sp1 and Sp3 by use of small interfering RNA (siRNA) significantly reduced the promoter activity. Binding of Sp1 and Sp3 to the promoter region was confirmed using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). Our data explain why certain HERV-K proviruses have lost promoter competence. Since vertebrate promoters lacking canonical core promoter elements are common but poorly studied, understanding the HERV-K promoter not only will provide insight into the regulation of endogenous retroviruses but also can serve as a paradigm for understanding the regulation of this class of cellular genes.