Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

Human endogenous retrovirus K (HML-2) RNA and protein expression is a marker for human embryonic and induced pluripotent stem cells.

BACKGROUND: Malignant human embryonal carcinoma cells (ECCs) rely on similar transcriptional networks as non-malignant embryonic stem cells (ESCs) to control selfrenewal, maintain pluripotency, and inhibit differentiation. Because re-activation of silenced HERV-K(HML-2) loci is a hallmark of ECCs, we asked if this HERV group was also reactivated in ESCs and induced pluripotent stem cells (iPSCs). FINDINGS: Using RT-PCR and Western Blot, we demonstrate HERV-K(HML-2) RNA and protein expression in undifferentiated human ESCs and iPSCs. Induction of differentiation by embryoid body formation resulted in rapid silencing of HERV-K(HML-2) provirus expression. Sequencing analysis of a conserved region of the gag gene showed that proviral expression in ESCs and iPSCs represents at least 11 of the 66 nearly full length HERV-K(HML-2) loci, with slightly varying patterns in individual cell lines. These proviruses are human specific integrations and harbor promoter competent long terminal repeats (LTR5hs subgroup). We observed high mRNA levels of the NP9 and Gag encoding proviruses K101(22q11.21) in all and K10(5q33.3) in most of the ECC, ESC, and iPSC lines tested, while K37(11q23.3) mRNA was detected only in ESCs and iPSCs. In addition, we detected expression of proviral mRNA encoding the RNA export adaptor Rec in all cell lines studied. Proviral mRNA originating from the K108(7p22.1) locus, which inter alia codes for functional Rec and Env proteins, was only reactivated in malignant ECC lines, not in benign ESCs or iPSCs. CONCLUSIONS: HERV-K(HML-2) RNA and protein expression is a marker for pluripotent human stem cells. Initiation of differentiation results in rapid down-regulation. Further studies are needed to explore a putative functional role of HERV-K(HML-2) RNA and proteins in pluripotent stem cells.

Vaccination directed against the human endogenous retrovirus-K envelope protein inhibits tumor growth in a murine model system.

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer.

Differential gene expression in ERα-positive and ERα-negative breast cancer cells upon leptin stimulation.

In postmenopausal women, adipositas represents a serious risk factor for cancer development and progression. White adipose tissue secretes the 16 kDa hormone leptin which plays a key role in the regulation of appetite and metabolism. An increasing number of reports indicate that leptin also interferes with signal transduction pathways implicated in the development of breast cancer. In our previous study, we identified the estrogen receptor alpha (ERα) as a relevant enhancer of leptin-induced signal transduction leading to transactivation of signal transducer and activator of transcription 3 (Stat3). The purpose of this study is the investigation of specific target gene expression in response to leptin-mediated Stat3 signaling. We performed a comprehensive microarray analysis of ERα-positive and ERα-negative MDA-MB-231 cells upon leptin treatment and identified 49 genes which showed a significant ERα-dependent regulation in leptin-treated MDA-MB-231 cells. There was no intersection with genes which were merely up- or downregulated by ERα expression and only 9 and 11 genes overlapping targets which were regulated by leptin stimulation either in ERα-expressing or ERα-negative MDA-MB-231 cells, respectively. To demonstrate the specificity, expression of three target genes was validated by quantitative real-time PCR. In conclusion, these data imply that leptin can induce a different set of target genes dependent on ERα expression, which might contribute to the development and progression of cancer diseases.

Biochemical analysis of the complex between the tetrameric export adapter protein Rec of HERV-K/HML-2 and the responsive RNA element RcRE pck30.

The RNA export adaptor protein Rec, encoded for by the human endogenous retrovirus HERV-K/HML-2 elements, binds to the Rec responsive element (RcRE) located in the 3' untranslated region of HERV-K/HML-2 transcripts. Binding allows the nucleocytoplasmic export of unspliced viral RNA, thereby overcoming host restriction. Chemical probing of the secondary structure of the RcRE corroborated the theory that the RcRE forms a complex folded structure with seven stem-loop regions. Laser-induced liquid beam ion desorption mass spectrometry revealed that Rec forms stable tetramers, which are further stabilized upon RNA binding. The RNA protein complex consists of three Rec tetramers, which bind to multiple sites on the RcRE-preferentially to purine-rich motifs-which represent several low-affinity binding sites. Mutated RcREs, with one to three purine-rich motifs deleted, were still bound and exported by Rec, indicating that the complex folded structure of the RcRE is important for Rec binding. This suggests a binding model where up to three Rec tetramers bind to the complex folded structure of the RcRE and the binding seems to be tightened by recognition of the purine-rich motifs.

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

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.

Expression of estrogen receptor alpha increases leptin-induced STAT3 activity in breast cancer cells.

Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ERalpha (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ERalpha. Downregulation of ERalpha using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ERalpha ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ERalpha ligands. We also detected ERalpha binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ERalpha-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ERalpha-dependent development of malignant diseases.

5'-Transducing SVA retrotransposon groups spread efficiently throughout the human genome.

SVA elements represent the youngest family of hominid non-LTR retrotransposons, which alter the human genome continuously. They stand out due to their organization as composite repetitive elements. To draw conclusions on the assembly process that led to the current organization of SVA elements and on their transcriptional regulation, we initiated our study by assessing differences in structures of the 116 SVA elements located on human chromosome 19. We classified SVA elements into seven structural variants, including novel variants like 3'-truncated elements and elements with 5'-flanking sequence transductions. We established a genome-wide inventory of 5'-transduced SVA elements encompassing approximately 8% of all human SVA elements. The diversity of 5' transduction events found indicates transcriptional control of their SVA source elements by a multitude of external cellular promoters in germ cells in the course of their evolution and suggests that SVA elements might be capable of acquiring 5' promoter sequences. Our data indicate that SVA-mediated 5' transduction events involve alternative RNA splicing at cryptic splice sites. We analyzed one remarkably successful human-specific SVA 5' transduction group in detail because it includes at least 32% of all SVA subfamily F members. An ancient retrotransposition event brought an SVA insertion under transcriptional control of the MAST2 gene promoter, giving rise to the primal source element of this group. Members of this group are currently transcribed. Here we show that SVA-mediated 5' transduction events lead to structural diversity of SVA elements and represent a novel source of genomic rearrangements contributing to genomic diversity.

Serological response to human endogenous retrovirus K in melanoma patients correlates with survival probability.

A few years ago, reactivation of human endogenous retrovirus K (HERV-K) proviruses in melanoma was described. The expression of HERV-K proteins induces humoral immune responses. The aim of the present study was to elucidate the prognostic relevance of serological anti-HERV-K reactivity in melanoma patients. In a retrospective study, anti-HERV-K Gag and Env antibodies were detected in 51 of the 312 randomly selected and blinded sera from melanoma patients, but not in any of the 70 sera from healthy controls. Comparing serological HERV-K reactivity with established melanoma markers revealed a significant correlation (p = 0.018, Chi-square test) with the stage of disease classified according to the American Joint Committee on Cancer (AJCC). Anti-HERV-K reactivity was elevated in patients with acrolentiginous/mucosal/uveal melanoma (tumor subtypes developing at sun-protected sites) compared to patients with lentigo/nodular/superficial spreading melanoma (p = 0.011, Chi-square test). Patients with anti-HERV-K antibodies had a significantly decreased disease-specific overall survival (stage I-IV, p < 0.001; stage I-III, p = 0.005, log-rank test). Significantly, multivariate Cox regression analysis including prognostic markers in clinical use (e.g., AJCC stage, T-class, serum level of S100-beta) revealed serological HERV-K reactivity as an independent marker of reduced survival probability (p = 0.027) in melanoma patients with the early stages of the disease (AJCC I-III). This is the first report that the humoral anti-HERV-K immune response may provide additional prognostic information to that of established melanoma markers.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines.

The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.

Identification of a melanoma marker derived from melanoma-associated endogenous retroviruses.

We previously described the expression of melanoma-associated endogenous retrovirus (MERV) proteins and viral particles in human melanomas and metastases. The objective of the present study was to determine whether a humoral immune response to MERV proteins occurs in melanoma. Candidate B-cell epitopes on MERV proteins were predicted using bioinformatic screening. The reactivity of MERV peptides corresponding to the predicted epitopes with antibodies prevalent in sera of melanoma patients was analyzed. An immunodominant peptide located in the env protein of MERV was identified. Subsequent analyzes using 81 samples from stage I to stage IV melanoma patients and 95 sera from healthy subjects revealed statistically significant differences in seroprevalence of antibodies in melanoma sera samples when compared with reference samples from healthy subjects. The prevalence of anti-MERV antibodies in melanoma patient sera was confirmed by immunofluorescence on env-transfected cells. These data indicate the potential of this candidate peptide as target for diagnosis and immunotherapy.

Leptin receptor isoform 219.1: an example of protein evolution by LINE-1-mediated human-specific retrotransposition of a coding SVA element.

Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

An endogenous retrovirus derived from human melanoma cells.

We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.