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Human endogenous retroviruses (HERVs) and mammalian apparent long terminal repeat (LTR) retrotransposons (MaLRs) are retroviral sequences that integrated into germ line cells millions of years ago. Transcripts of these LTR retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. Here, we focused on the HERV/MaLR expression and modulation in a scenario of immune system activation. We used a public data set of human peripheral blood mononuclear cells (PBMCs) RNA-Seq from 15 healthy participants to a clinical trial before and after exposure to lipopolysaccharide (LPS), for which we established an RNA-Seq workflow for the identification of expressed and modulated cellular genes and LTR retrotransposon elements. IMPORTANCE We described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4% of the LTR retrotransposon loci were expressed and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERV and MaLR loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci as differentially expressed, checking their genomic context of insertion and observing a general colocalization with genes that are involved and modulated in the immune response, as a consequence of LPS stimulation. The analyses of HERV and MaLR expression and modulation show that these LTR retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR retrotransposons and the immune host response., (Copyright © 2020 American Society for Microbiology.)

Telomere length maintenance is essential for most eukaryotes to ensure genome stability and integrity. A non-long terminal repeat (LTR) retrotransposon, SART1Bm, targets telomeric repeats (TTAGG)n of the silkworm Bombyx mori and is presumably involved in telomere length maintenance. However, how many telomeric repeats are required for its retrotransposition and how reverse transcription is initiated at the target site are not well understood. Here, using an ex vivo and trans-in vivo recombinant baculovirus retrotransposition system, we demonstrated that SART1Bm requires at least three (TTAGG) telomeric repeats and a longer poly(A) tail for its accurate retrotransposition. We found that SART1Bm retrotransposed only in the third (TTAGG) tract of three repeats and that the A residue of the (TTAGG) unit was essential for its retrotransposition. Interestingly, SART1Bm also retrotransposed into telomeric repeats of other species, such as human (TTAGGG)n repeats, albeit with low retrotransposition efficiency. We further showed that the reverse transcription of SART1Bm occurred inaccurately at the internal site of the 3' untranslated region (UTR) when using a short poly(A) tail but at the accurate site when using a longer poly(A) tail. These findings promote our understanding of the general mechanisms of site-specific retrotransposition and aid the development of a site-specific gene knock-in tool.

Overexpression and long terminal repeat (LTR) polymorphism of the HRES‑1/Rab4 human endogenous retrovirus locus have been associated with T cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is diminished overall in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus affect T cell activation. The results showed that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR enhancer are hypomethylated in SLE. Pharmacologic or genetic inactivation of DNA methyltransferase 1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR enhancer and exerted control over HRES-1/Rab4 expression in rs451401 genotype- and methylation-dependent manners. The LTR enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation- and rs451401 allele-dependent transducer of environmental stress and controller of T cell activation.

DNA methylation and histone modifications critically regulate the expression of many genes and repeat regions during spermatogenesis. However, the molecular details of these processes in male germ cells remain to be addressed. Here, using isolated murine sperm cells, ultra-low-input native ChIP-Seq (ULI-NChIP-Seq), and whole genome bisulfite sequencing (WGBS), we investigated genome-wide DNA methylation patterns and histone 3 Lys-9 trimethylation (H3K9me3) modifications during mouse spermatogenesis. We found that DNA methylation and H3K9me3 have distinct sequence preferences and dynamics in promoters and repeat elements during spermatogenesis. H3K9me3 modifications in histones at gene promoters were highly enriched in round spermatids. H3K9me3 modification on long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs) was involved in silencing active transcription from these regions in conjunction with reestablishment of DNA methylation. Furthermore, H3K9me3 remodeling on the X chromosome was involved in meiotic sex chromosome inactivation and in partial transcriptional reactivation of sex chromosomes in spermatids. Our findings also revealed the DNA methylation patterns and H3K9me3 modification profiles of paternal and maternal germline imprinting control regions (gICRs) during spermatogenesis. Taken together, our results provide a genome-wide map of H3K9me3 modifications during mouse spermatogenesis that may be helpful for understanding male reproductive disorders., (© 2019 Liu et al.)

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats ( LTRs ), gag , pol , and env . Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells., Competing Interests: The authors declare no conflicts of interest., (© 2019 The Korean Society of Veterinary Science.)

Frequent somatic variations exist in pitaya (Hylocereus undatus) plants grown under abiotic stress conditions. Long terminal repeat (LTR) retrotransposons can be activated under stressful conditions and play key roles in plant genetic variation and evolution. However, whether LTR retrotransposons promotes pitaya somatic variations by regulating abiotic stress responses is still uncertain. In this study, transcriptionally active LTR retrotransposons were identified in pitaya after exposure to a number of stress factors, including in vitro culturing, osmotic changes, extreme temperatures and hormone treatments. In total, 26 LTR retrotransposon reverse transcriptase (RT) cDNA sequences were isolated and identified as belonging to 9 Ty1-copia and 4 Ty3-gypsy families. Several RT cDNA sequences had differing similarity levels with RTs from pitaya genomic DNA and other plant species, and were differentially expressed in pitaya under various stress conditions. LTR retrotransposons accounted for at least 13.07% of the pitaya genome. HuTy1P4 had a high copy number and low expression level in young stems of pitaya, and its expression level increased after exposure to hormones and abiotic stresses, including in vitro culturing, osmotic changes, cold and heat. HuTy1P4 may have been subjected to diverse transposon events in 13 pitaya plantlets successively subcultured for four cycles. Thus, the expression levels of these retrotransposons in pitaya were associated with stress responses and may be involved in the occurrence of the somaclonal variation in pitaya., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Retrotransposons constitute a large portion of plant genomes. The chromosomal distribution of a wide variety of retrotransposons has been analyzed using genome sequencing data in several plants, but the evolutionary profile of transposition has been characterized for a limited number of retrotransposon families. Here, we characterized 96 elements of the SORE-1 family of soybean retrotransposons using genome sequencing data. Insertion time of each SORE-1 element into the genome was estimated on the basis of sequence differences between the 5' and 3' long terminal repeats (LTRs). Combining this estimation with information on the chromosomal location of these elements, we found that the insertion of the existing SORE-1 into gene-rich chromosome arms occurred on average more recently than that into gene-poor pericentromeric regions. In addition, both the number of insertions and the proportion of insertions into chromosome arms profoundly increased after 1 million years ago. Solo LTRs were detected in these regions at a similar frequency, suggesting that elimination of SORE-1 via unequal homologous recombination was unbiased. Taken together, these results suggest the preference of a recent insertion of SORE-1 into chromosome arms comprising euchromatic regions. This notion is contrary to an earlier view deduced from an overall profiling of soybean retrotransposons and suggests that the pattern of chromosomal distribution can be more diverse than previously thought between different families of retrotransposons.

LTR retrotransposons are major components of plant genomes. They are regulated by a diverse array of external stresses and tissue culture conditions, displaying finely tuned responses to these stimuli, mostly in the form of upregulation. Second to stress conditions and tissue culture, meristems are also permissive for LTR retrotransposon expression, suggesting that a dedifferentiated cell status may represent a frequent activating condition. LTR regions are highly plastic and contain regulatory motifs similar to those of cellular genes. The activation of LTR retrotransposons results from interplay between the release of epigenetic silencing and the recruitment by LTRs of specific regulatory factors. Despite the role of LTR retrotransposons in driving plant genome diversification, convincing evidence for major mobilizations of LTR retrotransposons remains much rarer than observations of massive bursts of transcriptional upregulation. Current evidence suggests that LTR retrotransposon expression may be involved in host functional plasticity, acting as dispersed regulatory modules able to redirect stress stimuli to adjacent plant genes. This may be of crucial importance for plants that cannot escape stress, and have evolved complex and highly coordinated responses to external challenges. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.

We have previously shown that TPA activates HTLV-1 LTR in Jurkat T-cells by inducing the binding of Sp1-p53 complex to the Sp1 site residing within the Ets responsive region 1 (ERR-1) of the LTR and that this activation is inhibited by PKCalpha and PKCepsilon. However, in H9 T-cells TPA has been noted to activate the LTR in two consecutive stages. The first stage is activation is mediated by PKCetta and requires the three 21 bp TRE repeats. The second activation mode resembles that of Jurkat cells, except that it is inhibited by PKCdelta. The present study revealed that the first LTR activation in H9 cells resulted from PKCetta-induced elevation of non-phosphorylated c-Jun which bound to the AP-1 site residing within each TRE. In contrast, this TRE-dependent activation did not occur in Jurkat cells, since there was no elevation of non-phosphorylated c-Jun in these cells. However, we found that PKCalpha and PKCepsilon, in Jurkat cells, and PKCetta and PKCdelta, in H9 cells, increased the level of phosphorylated c-Jun that interacted with the Sp1-p53 complex. This interaction prevented the Sp1-p53 binding to ERR-1 and blocked, thereby, the ERR-1-mediated LTR activation. Therefore, this PKC-inhibited LTR activation started in both cell types after depletion of the relevant PKCs by their downregulation. In view of these variable activating mechanisms we assume that there might be additional undiscovered yet modes of HTLV-1 LTR activation which vary in different cell types. Moreover, in line with this presumption we speculate that in HTLV-1 carriers the LTR of the latent provirus may also be reactivated by different mechanisms that vary between its different host T-lymphocyte subclones. Since this reactivation may initiate the ATL process, understanding of these mechanisms is essential for establishing strategies to block the possibility of reactivating the latent virus as preventive means for ATL development in carriers.

The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates DNA replication of terminal repeat (TR) DNA to enable viral episome persistence in latently infected cells. Southern blotting is routinely used to detect LANA-replicated DNA. We developed and validated a real-time PCR assay for TR-associated DNA and compared it with Southern blot analysis. Both PCR and Southern blot detected LANA-replicated DNA, but the PCR assay was more rapid and did not require radioisotope. PCR detection at 24 and 72 hours post-transfection demonstrated rapid loss of transfected TR DNA. LANA, and to a lesser extent a moderately deficient LANA mutant, reduced the rate of DNA loss through addition of replicated TR DNA and reduction in the loss of non-replicated DNA, the latter of which is consistent with LANA's nuclear segregation function. Therefore, this work develops a rapid, sensitive, and quantitative PCR (qPCR) assay to detect KSHV LANA-replicated DNA and demonstrates that LANA reduces TR DNA loss after transfection through replication and nuclear partitioning of TR DNA.

It is now commonly agreed that the human genome is not the stable entity originally presumed. Deletions, duplications, inversions, and insertions are common, and contribute significantly to genomic structural variations (SVs). Their collective impact generates much of the inter-individual genomic diversity observed among humans. Not only do these variations change the structure of the genome; they may also have functional implications, e.g. altered gene expression. Some SVs have been identified as the cause of genetic disorders, including cancer predisposition. Cancer cells are notorious for their genomic instability, and often show genomic rearrangements at the microscopic and submicroscopic level to which transposable elements (TEs) contribute. Here, we review the role of TEs in genome instability, with particular focus on non-LTR retrotransposons. Currently, three non-LTR retrotransposon families - long interspersed element 1 (L1), SVA (short interspersed element (SINE-R), variable number of tandem repeats (VNTR), and Alu), and Alu (a SINE) elements - mobilize in the human genome, and cause genomic instability through both insertion- and post-insertion-based mutagenesis. Due to the abundance and high sequence identity of TEs, they frequently mislead the homologous recombination repair pathway into non-allelic homologous recombination, causing deletions, duplications, and inversions. While less comprehensively studied, non-LTR retrotransposon insertions and TE-mediated rearrangements are probably more common in cancer cells than in healthy tissue. This may be at least partially attributed to the commonly seen global hypomethylation as well as general epigenetic dysfunction of cancer cells. Where possible, we provide examples that impact cancer predisposition and/or development., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Malignancy results from a complex combination of genetic and epigenetic changes, the full effects of which are still largely unknown. Here we summarize current knowledge of the origin, retrotranspositional activity, epigenetic state, and transcription of human endogenous retroviruses (HERVs), and then discuss the potential effects of their deregulation in cancer. Evidence suggests that cancer-associated epigenetic changes most likely underlie potential HERV-mediated effects on genome and transcriptome instability and may play a role in malignancy. Despite our currently limited understanding of the importance of HERVs or other transposable elements in cancer development, we believe that the emerging era of high-throughput sequencing of cancer genomes, epigenomes, and transcriptomes will provide unprecedented opportunities to investigate these roles in the future., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Long terminal repeats (LTRs) of human endogenous retrovihuses (HERs) might affect transcription regulation of neighboring genes. In our previous study, we showed that the solitary LTR residing in the KIAA1245/NBPF gene subfamily displayed high enhancer activity in a transformed embryonal carcinoma cell line Tera1. In this study, we performed a functional dissection of the LTR and studied its deletion series. Using transient transfection assay, we confirmed the ability of the LTR to drive the expression of the luciferase reporter gene in Teral cells. At the same time, in two other transformed cell lines tested, NGP and NT2/D1, the full-size LTR and its fragments showed no or low enhancer activity, thus demonstrating cell type specificity of the LTR enhancer activity. The functional dissection of the LTRrevealed a specific region within the U3 part appeared to be responsible for the enhancer properties. We showed that the identified enhancer was able to work in a highly cell type specific manner. The data obtained are in line with the hypothesis suggesting that KIAA1245/NBPF LTR may affect the regulation of the KIAA1245/NBPF subfamily genes transcription.

Recently, the first human infection with an exogenous gammaretrovirus (XMRV) was reported. In its initial description, XMRV was confined to prostate stromal fibroblasts, although subsequent reports demonstrated XMRV protein expression in prostate epithelial cells. Most recently, XMRV has been detected in blood cells of patients with chronic fatigue syndrome. The aim of this study was to elucidate the transmission routes and tissue tropism of XMRV by comparing its host range, receptor usage and LTR functionality with other MLV isolates. We demonstrate using pseudotype experiments that XMRV Env mediates efficient infection of cells from different species. We show that replication competent XMRV infects various human cell types, including hematopoietic cell lines and prostate stromal fibroblasts. XMRV-LTR activity is significantly higher in the prostate cancer cell line LNCaP and in prostate stromal fibroblasts, compared to other cell types tested and could be one factor contributing to efficient viral spread in prostate tissue., (Copyright 2009 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in alpha-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a gamma-retroviral vector (gamma-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) gamma-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective. This report demonstrates that intravenous (i.v.) injection of a SIN gamma-RV expressing canine IDUA from the liver-specific human alpha(1)-antitrypsin promoter into adult or newborn MPS I mice completely prevents biochemical abnormalities in several organs, and improved bone disease, vision, hearing, and aorta to a similar extent as was seen with administration of the LTR-intact vector to adults. Improvements were less profound than when using an LTR-intact gamma-RV in newborns, which likely reflects a lower level of transduction and expression for the SIN vector-transduced mice, and might be overcome by using a higher dose of SIN vector. A SIN gamma-RV vector ameliorates clinical manifestations of MPS I in mice and should be safer than an LTR-intact gamma-RV.

CCAAT/enhancer-binding protein (C/EBP) beta and C/EBP sites in the HIV-1 long terminal repeat (LTR) are crucial for HIV-1 replication in monocyte/macrophages and for the ability of interferon beta (IFN beta) to inhibit ongoing active HIV replication in these cells. This IFN beta-mediated down-regulation involves induction of the truncated, dominant-negative isoform of C/EBP beta referred to as liver-enriched transcriptional inhibitory protein (LIP). Although binding of the C/EBP beta isoform to C/EBP sites in the simian immunodeficiency virus (SIV) LTR has previously been examined, the importance of these sites in core promoter-mediated transcription, virus replication, IFN beta-mediated regulation, and the relative binding of the two isoforms (C/EBP beta and LIP) has not been investigated. Here, we specifically examine two C/EBP sites, JC1 (-100 bp) and DS1 (+134 bp), located within the minimal region of the SIV LTR, required for core promoter-mediated transcription and virus replication in macrophages. Our studies revealed that the JC1 but not DS1 C/EBP site is important for basal level transcription, whereas the DS1 C/EBP site is imperative for productive virus replication in primary macrophages. In contrast, either JC1 or DS1 C/EBP site is sufficient to mediate IFN beta-induced down-regulation of SIV LTR activity and virus replication in these cells. We also characterized the differential binding properties of C/EBP beta and LIP to the JC1 and DS1 sites. In conjunction with previous studies from our laboratory, we demonstrate the importance of these sites in virus gene expression, and we propose a model for their role in establishing latency and persistence in macrophages in the brain.

Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

Insertional mutagenesis leading to insurgence of leukemia has been shown as a consequence of retroviral (RV)-mediated gene transfer in animal models and in clinical trials of gene therapy for X-linked severe combined immunodeficiency. Aberrant expression of oncogenes neighboring the gamma-RV vector insertion site via induction by the enhancer element of the viral long terminal repeats (LTRs) is thought to have played a role in leukemogenesis. Consequently, RV vectors devoid of LTR enhancer elements could prove as safer tools for gene transfer. To test this hypothesis, we evaluated the immortalization ability of two RV vectors: one carrying the full-length Moloney leukemia virus (MLV) LTR and one with the same LTR in which the enhancer element was deleted [MLV self-inactivating (SIN)]. Unexpectedly, transduction with MLV SIN resulted in an only slightly and not significant decreased immortalization frequency of primary bone marrow (BM) cultures (about 37%) compared to transduction with MLV (about 48%). Similar to MLV, immortalization by MLV SIN is likely caused by insertional activation of oncogenes including Evi1, Mds1, Mef2c, and Hoxa7. Our results indicate that the MLV SIN, devoid of the LTR enhancer element, was still able to immortalize BM cells by activating nearby gene expression, indicating the need of an accurate selection of the internal promoter to obtain safer SIN RV vectors.

Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3' long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3' long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3'-end of the mouse mammary tumor virus genome, but further deletions at the 5'- or 3'-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3'-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5'- and 3'-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

Using a model system in which the expression of the reporter gene lacZ is under the control of five deleted variants of the copia retrotransposon regulatory region, which includes the 5'-long terminal repeat (LTR) and the 5'-untranslated region (5'-UTR), their contribution to the control of retrotransposon activity in different organs of males and females of Drosophila melanogaster was analyzed. The whole regulatory region provides expression of the reporter gene at the embryonic stage, and in larvae and adult flies only in generative organs. The 5'-end of LTR harbors a positive regulator that determines expression of the retrotransposon in organs of all types. The 3'-end of LTR harbors a negative regulator, which is sex- and time-specific: it represses copia expression in generative organs of males at all stages of development, and only at the imaginal stage in somatic tissues, without any effect on the expression of the retrotransposon in females. 5'-UTR contains a negative regulator of copia expression: it decreases the expression in embryos and generative organs and blocks it in somatic tissues. It may be suggested that a complex set of regulatory elements was formed in the course of the evolution of the retrotransposon, which made it possible to maintain a certain level of its expression in different types of cells and tissues and at different stages of development and, thus, to limit the harm caused to the host and provide the possibility for the retrotransposon to exist in the host genome over many generations.

Transcription of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is activated through binding of the viral Tat protein to the trans-activating response (TAR) element at the 5' end of the nascent transcript. Whereas HIV type 1 (HIV-1) TAR folds a simple hairpin structure, the corresponding domains of HIV-2 and SIVmac exhibit a more complex structure composed of three stem-loops. This structural polymorphism may be attributed to additional functions of TAR in HIV-2/SIVmac replication. We recently constructed an SIVmac variant that does not require the Tat-TAR interaction for transcription. We used this variant to study additional roles of TAR in SIVmac replication and generated mutants with a truncated TAR structure. We demonstrate that partial or nearly complete removal of TAR does not impair viral transcription, RNA processing, and translation. Moreover, these deletions do not significantly affect virus replication in the PM1 T-cell line and macaque peripheral blood mononuclear cells. These results demonstrate that the complex TAR structure in SIVmac has no other essential function in virus replication in vitro besides its role in Tat-mediated activation of transcription.

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Tangles containing hyperphosphorylated aggregates of insoluble tau are a pathological hallmark of progressive supranuclear palsy (PSP). Several phosphorylation sites on tau in PSP have been identified using phospho-specific antibodies, but no sites have been determined by direct sequencing due to the difficulty in enriching insoluble tau from PSP brain. We describe a new method to enrich insoluble PSP-tau and report eight phosphorylation sites [Ser46, Thr181, Ser202, Thr217, Thr231, Ser235, Ser396/Ser400 (one site) and Thr403/Ser404 (one site)] identified by mass spectrometry. We also describe a 35 kDa C-terminal tau fragment (tau35), lacking the N-terminus of tau but containing four microtubule-binding repeats (4R), that is present only in neurodegenerative disorders in which 4R tau is over-represented. Tau35 was readily detectable in PSP, corticobasal degeneration and 4R forms of fronto-temporal dementia with parkinsonism linked to chromosome 17, but was absent from control, Alzheimer's disease and Pick's disease brain. Our findings suggest the aggregatory characteristics of PSP-tau differ from those of insoluble tau in Alzheimer's disease brain and this might be related to the presence of a C-terminal cleavage product of tau.

In this work the structural variations of Terminal Inverted Repeats (TIR) of Bari like transposons in Drosophila species has been studied. The aim is to try and assess the relevance of different variants in the evolutionary distribution of Bari elements. Bari is a member of the widespread Tc1 superfamily of transposable elements that has colonized most species of the Drosophila genus. We previously reported the structure of two related elements that differ in their TIR organization: Bari1 harbouring 26-bp TIR (short TIRs) and Bari2 with about 250-bp TIR (long TIIR). While elements with short TIRs are complete and potentially autonomous, long ones are invariably composed of defective copies. The results show that in D. pseudobscura, D. persimilis and D. mojavensis, there is a third class of Bari elements, Bari3, that exhibit a long TIR structure and are not defective. Phylogenetic relationships among reconstructed transposases are consistent with the three subfamilies sharing a common origin. However, the final TIR organization into long or short structure is not related by descent but appears to be lineage-specific. Furthermore, we show that, independently of origin and organization, within the 250-bp terminal sequences there are three regions that are conserved in both sequence and position suggesting they are under functional constraint.

Metnase, also known as SETMAR, is a SET and transposase fusion protein with an undefined role in mammalian DNA repair. The SET domain is responsible for histone lysine methyltransferase activity at histone 3 K4 and K36, whereas the transposase domain possesses 5'-terminal inverted repeat (TIR)-specific DNA binding, DNA looping, and DNA cleavage activities. Although the transposase domain is essential for Metnase function in DNA repair, it is not clear how a protein with sequence-specific DNA binding activity plays a role in DNA repair. Here, we show that human homolog of the ScPSO4/PRP19 (hPso4) forms a stable complex with Metnase on both TIR and non-TIR DNA. The transposase domain essential for Metnase-TIR interaction is not sufficient for its interaction with non-TIR DNA in the presence of hPso4. In vivo, hPso4 is induced and co-localized with Metnase following ionizing radiation treatment. Cells treated with hPso4-siRNA failed to show Metnase localization at DSB sites and Metnase-mediated stimulation of DNA end joining coupled to genomic integration, suggesting that hPso4 is necessary to bring Metnase to the DSB sites for its function(s) in DNA repair.

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC.

By associating with cyclic AMP-responsive element-binding protein (CREB), the human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates transcription from the HTLV-1 long terminal repeat (LTR), which contains multiple cyclic AMP-responsive elements. The transducers of regulated CREB activity (TORCs) were a recently identified family of CREB co-activators that bind to CREB to enhance CRE-mediated transcription. TORC3, a TORC family protein, dramatically enhances Tax-mediated transcription from the LTR. In this study, we performed a yeast two-hybrid screen using the N-terminal region of TORC3 as bait and identified B-cell chronic lymphatic leukemia protein 3 (BCL3) as a protein interacting with TORC3. This interaction was confirmed by glutathione S-transferase pulldown assays and co-immunoprecipitation experiments with detection by Western blotting. The ankyrin repeat domain of BCL3 interacted with TORC3. By using a luciferase assay, we determined that BCL3 inhibited transcription from the HTLV-1 LTR in a manner dependent on TORC3. Knockdown of endogenous BCL3 using RNA interference enhanced transcriptional activation of CRE. Treatment with trichostatin A, a potent inhibitor of the transcriptional co-repressor HDAC, partially reversed the inhibitory effect of BCL3. These results suggest that BCL3 functions as a repressor of HTLV-1 LTR-mediated transcription through interactions with TORC3. In addition to stimulating transcription from the HTLV-1 LTR, Tax also enhances BCL3 expression; thus, transcription from the LTR is regulated by both positive and negative feedback mechanisms.

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-end-joining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.

LTR retrotransposons (LTR-RTNs) are widespread constituents of eukaryote genomes, particularly plant genomes. Although LTR-RTNs from plants were thought to be transcriptionally silent in somatic tissues, evidences of activity under certain conditions are available for some of them. In order to investigate LTR-RTNs in the Citrus sinensis genome, we analysed them by PCR using degenerate primers corresponding to highly conserved domains. All elements of the two types of LTR-RTN comprise about 23% of the genome, the copia group contribution being higher (13%) than the gypsy one (10%). From dendogram analysis, we report seven new copia RTN families, named CIRE1 to CIRE7. Here, we report on the first complete retrotransposon identified in Citrus (named CIRE1), which has all the features of a typical copia RTN. CIRE1 retrotransposon has around 2,200 full-length copies, contributing to 2.9% of the C. sinensis genome. CIRE1 has a root-specific expression in sweet orange plants. We have also determined that wounding and exogenous application of plant hormones, as methyl jasmonate and auxin, increase the transcription level of CIRE1 in leaf tissues. In addition, we show that CIRE1 5'LTR promoter can drive transient expression of the gus reporter gene in heterologous plant systems. These findings confirm CIRE1 as one of the few transcriptionally active RTNs described in plants and to our knowledge the first one to be reported in Citrus species.

Eukaryotic genomes contain vast amounts of repetitive DNA derived from transposable elements (TEs). Large-scale sequencing of these genomes has produced an unprecedented wealth of information about the origin, diversity, and genomic impact of what was once thought to be "junk DNA." This has also led to the identification of two new classes of DNA transposons, Helitrons and Polintons, as well as several new superfamilies and thousands of new families. TEs are evolutionary precursors of many genes, including RAG1, which plays a role in the vertebrate immune system. They are also the driving force in the evolution of epigenetic regulation and have a long-term impact on genomic stability and evolution. Remnants of TEs appear to be overrepresented in transcription regulatory modules and other regions conserved among distantly related species, which may have implications for our understanding of their impact on speciation.

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

Jembrana disease virus (JDV) is a lentivirus highly related to the bovine immunodeficiency virus (BIV). It causes an acute disease with high mortality rate within 1-2 weeks. JDV encodes the most potent Tat (JTat) of any of the lentiviruses. JTat can transactivate all LTRs and functionally substitute for HIV Tat in the viral genome and may function as a pivotal regulator in the acute pathogenesis of JDV. The goal of this paper is to study JTat internalization by cells, the mechanisms involved in internalization, and the effect of JTat on neighbouring cells. By quantification and fluorescence microscopy, we found that the internalization of extracellular EGFP-JTat fusion protein was both time and dose-dependent, but endocytosis and energy independent. We identified that arginines which were responsible for the internalization. Internalized JTat was distributed in both the nucleus and the cytoplasm, could transactivate JDV LTR and modulate cellular gene expression. Based on our findings, we propose that secretion and internalization of JTat may be a way for JDV to influence neighbouring cells and make the cellular environment more amenable to viral infection.

Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30(II)-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30(II)-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30II modulates viral gene expression in a cooperative manner with p300-mediated acetylation.

Human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription from the long terminal repeats (LTR). Mechanisms through which Tax activates LTR have been established, but coactivators of this process remain to be identified and characterized. Here we show that all three members of the TORC family of transcriptional regulators are coactivators of Tax for LTR-driven expression. TORC coactivation requires CREB, but not ATF4 or other bZIP factors. Tax physically interacts with TORC1, TORC2, and TORC3 (TORC1/2/3), and the depletion of TORC1/2/3 inhibited Tax activity. TORC coactivation can be further enhanced by transcriptional coactivator p300. In addition, coactivators in the p300 family are required for full activity of Tax independently of TORC1/2/3. Thus, both TORC and p300 families of coactivators are essential for optimal activation of HTLV-1 transcription by Tax.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep that arises from type II pneumocytes and Clara cells of the lung epithelium. Studies of the tropism of this virus have been hindered by the lack of an efficient system for viral replication in tissue culture. To map regulatory regions important for transcriptional activation, an in vivo footprinting method that couples dimethyl sulfate treatment and ligation-mediated PCR was performed in murine type II pneumocyte-derived MLE-15 cells infected with a chimeric Moloney murine leukemia virus driven by the JSRV enhancers (DeltaMo+JS Mo-MuLV). In vivo footprints were found in the JSRV enhancers in two regions previously shown to be important for JSRV long terminal repeat (LTR) activity: a binding site for the lung-specific transcription factor HNF-3beta and an E-box element in the distal enhancer adjacent to an NF-kappaB-like binding site. In addition, in vivo footprints were detected in two downstream motifs likely to bind C/EBP and NF-I. Mutational analysis of a JSRV LTR reporter construct (pJS21luc) revealed that the C/EBP binding site is critical for LTR activity, while the putative NF-I binding element is less important; elimination of these sites resulted in 70% and 40% drops in LTR activity, respectively. Electrophoretic mobility shift assays using nuclear extracts from MLE-15 murine Clara cell-derived mtCC1-2 cells with probes corresponding to the NF-I or C/EBP sites revealed several complexes. Antiserum directed against NF-IA, C/EBPalpha, or C/EBPbeta supershifted the corresponding protein-DNA complexes, indicating that these isoforms, which are also important for the expression of several cellular lung-specific genes, may be important for JSRV expression in lung epithelial cells.

Transcriptional activation of gene expression in HIV-1 is controlled by the interaction of sequence-specific transcription factors with the long terminal repeat (LTR) of the provirus. The identification and characterization of cellular proteins involved in the process has provided a basic understanding about both general eukaryotic and HIV-1 proviral transcription regulation. The HIV-1 epidemic is expanding worldwide with an increasing number of distinct viral subtypes as well as intersubtype recombinant viruses. LTR-specific sequence variability among different HIV-1 variants could affect LTR binding to cellular and/or viral factors, influencing the extent of transcription. In vitro assays have demonstrated subtype-specific functional differences between the LTR regions of distinct HIV-1 subtypes. This observation could have consequences on the biology of the different HIV-1 clades and influence HIV-1 disease progression. Finally, the knowledge of the molecular mechanisms of transcription regulation events could help in the search for new compounds targeting the critical steps of viral transcription.

R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.

The long terminal repeats (LTR) sequence divergence among Maedi Visna virus (MVV) isolates leads to LTRs with distinct transcriptional activities, which may result in distinct biological behaviours. The genetic heterogeneity, as well as basal and Tat-induced transcriptional activity of the LTRs from P1OLV and WLC-1 MVV viruses, slow/low and rapid/high isolates, respectively, have been examined and compared with LTRs from other strains of small ruminant lentiviruses (SRLV). Transfection assays using a reporter construct containing the LTR fused to a luciferase gene demonstrated that the LTR from P1OLV virus had the weakest promoter activity, suggesting a correlation between the level of promoter activity and the viral replication rate. To confirm this hypothesis, the promoter of P1OLV was cloned into infectious molecular clone KV1772kv72/67 and the resulting chimeric virus was tested for growth in various cell types. Compared to the parental KV1772, the LTR-chimeric virus KV1772/P1OLV exhibited a drastic reduction in replication rate in sheep choroid plexus (SCP) and lung cells, while in ovine macrophages and goat synovial membrane cells (GSM), chimeric virus showed a growth rate similar to that of parental virus. These observations suggest that the LTR is responsible for the slow/low in vitro phenotype presented by P1OLV in SCP and lung cells.

Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6-8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.

Friend murine leukemia virus (Fr-MLV) clone A8 causes thymoma 7 weeks postinfection in rats with a more rapid progression than clone 57. The U3 region of A8-LTR contains a unique structure of enhancer motifs consisting of three repeats of a 38-bp sequence containing FVa, LVb/C4, and CORE motifs. Replacement or deletion of the 38-bp sequence in the A8-U3 resulted in a marked reduction in tumorigenicity. Furthermore, the virus with 57-U3 gained high tumorigenicity after construction of the three 38-bp repeats in the U3 region. These findings indicated that the repeats of the 38-bp sequence of A8-LTR are essential for the rapid induction of thymoma. Interestingly, the repeat of the 38-bp sequence did not accelerate the amount of integrated viral DNA in the thymus during the early phase of infection, although it contributed to higher production of infectious virus. Thus, it was demonstrated that the ability to induce thymoma, which correlates with virus titer in the thymus, is not determined by the rate of viral DNA integration into the host genome.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.