Roswitha Löwer

The pathogenic potential of endogenous retroviruses: facts and fantasies

Endogenous retroviruses are descendants of viruses that became cellular genes by integration into their hosts genome. They still contribute to pathogenicity as a partner in recombination events, by de novo insertion after mobilization followed by activation of downstream proto-oncogenes, or by gene disruption. Re-expression of viral proteins accompanied by loss of immune tolerance could induce immune disturbances.

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.

HERV-K : The biologically most active human endogenous retrovirus family

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.

Human Teratocarcinomas Cultured in vitro Produce Unique Retrovirus-like Viruses

We have previously reported that among a series of human tumours investigated, only human teratocarcinoma cell lines derived from testicular tumours or pulmonary metastases of patients in Germany and the U.S.A. produced retrovirus-like particles spontaneously, albeit in low amounts. In a recent publication electron microscopical data suggested that the human teratocarcinoma-derived ( HTD ) particles were morphologically closely related, but not identical, to the type C retroviruses of animals. In this communication, the explantation of three human teratocarcinoma cell lines is briefly described. Evidence is presented that HTD particles (i) are synthesized only in a fraction of the epithelioid and differentiating cells; (ii) can be induced biochemically in a manner characteristic of retroviruses; (iii) either are not infectious or possess a peculiar host range; (iv) are immunologically unrelated to animal retrovirus strains; (v) possess an endogenous RNA-dependent DNA polymerase activity that can be banded at 1.16 g/ml in linear sucrose gradients. These results may be taken as suggestive evidence that HTD particles represent a novel group of unique retroviruses.

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.

Development of Insulin-Dependent Diabetes Mellitus Does Not Depend on Specific Expression of the Human Endogenous Retrovirus HERV-K

Conrad et al. stated that the sequence of the truncated IDDMK1,222 env gene encoded a superantigen. Sequence comparison with a variety of additional HTDV/HERV-K env sequences revealed extensive homologies with only a few nucleic acid substitutions. The superantigen sequences encoded by mouse mammary tumor virus (MMTV) also differ between virus strains, in particular within the COOH-terminal residues that may contribute to their Vβ specificity.As the IDDMK1,222 expression clone was not available to test for superantigen function, we amplified and cloned an HTDV/HERV-K sequence using a 5′ primer corresponding to the IDDMK1,222 superantigen sequence and the 3′ primer R-poly(A) described by Conrad et al. As the 3′ R-poly(A) primer was not RNA specific, we used genomic DNA as the template. The Env sequence obtained differed from IDDMK1,222 by six amino acids. One exchange abrogated the premature Env termination signal leading to a longer putative superantigen protein; the other changes were G46→V, H48→R, T115→P, G123→E, and V140→A. Preliminary attempts to use the THP1 cell line for expression of the Env protein failed due to insufficient transfection efficiency (less than 1% transfectants with a reporter gene using different transfection methods). We therefore used a well-established murine system (Gunzburg et al. 1993xGunzburg, W.H., Heinemann, F., Wintersberger, S., Miethke, T., Wagner, H., Erfle, V., and Salmons, B. Nature. 1993; 364: 154–158Crossref | PubMed | Scopus (27)See all ReferencesGunzburg et al. 1993) that allows for higher transfection efficiencies (30%–40% using electroporation). There is as yet no evidence that superantigens are species specific and, indeed, Conrad et al. recently reported similar results using human and murine test systems (Symposium on “Retroviruses and Autoimmunity,” 1998, London). However, murine A20 cells transiently transfected with the IDDMK1,222-related expression clone did not stimulate corresponding BALB/c T cells in a Vβ-specific fashion while A20 cells transfected with an MMTV superantigen induced a Vβ14-selective expansion of T cells. Truncating the longer Env sequence to the size of IDDMK1,222 did not alter the negative result.Clearly, further studies including more HTDV/HERV-K sequences as well as the expression clone established by Conrad et al. are needed to either confirm or refute the hypothesis that a superantigen is encoded by this HERV family. If the superantigen exists, its involvement in the onset or progression of insulin-dependent diabetes mellitus needs to be examined. With regard to other bacterial and especially viral superantigens, it seems very unlikely that the sequence published by Conrad et al. would be unique amongst all HTDV/HERV-K proviruses in encoding a superantigen.

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 ERα (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 ERα. Downregulation of ERα using small interfering RNA abolished leptin‐induced STAT3 phosphorylation. Interestingly, leptin‐mediated STAT3 activation was unaffected by co‐stimulation with the ERα ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin‐mediated STAT3 activity is independent of ERα ligands. We also detected ERα 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 ERα‐dependent cell viability. Altogether, our results indicate that leptin‐induced STAT3 activation acts as a key event in ERα‐dependent development of malignant diseases.

Rec (Formerly Corf) Function Requires Interaction with a Complex, Folded RNA Structure within Its Responsive Element rather than Binding to a Discrete Specific Binding Site

ABSTRACT It was recently reported that the human endogenous retrovirus HTDV/HERV-K encodes the regulatory protein Rec (formerly designated Corf), which is functionally equivalent to the nuclear export adapter proteins Rev of human immunodeficiency virus and Rex of human T-cell leukemia virus. We have demonstrated that the Rec protein interacts with a characteristic 429-nucleotide RNA element, the Rec-responsive element (RcRE), present in the 3′ long terminal repeat of HTDV/HERV-K transcripts. In analogy to the Rev and Rex proteins, which have distinct RNA binding sites in their responsive elements, we have proposed that Rec may also have a defined binding site in the RcRE. In this report, we demonstrate that not every HTDV/HERV-K copy present in the human genome contains an active RcRE, and we characterize mutations that abrogate Rec function. In addition, we demonstrate that Rec function requires binding to a complex, folded RNA structure rather than binding to a discrete specific binding site, in contrast to Rev and Rex and their homologous responsive elements. We define four stem-loop structures in the RcRE that are essential for Rec function. Finally, we demonstrate that both Rev and Rex can mediate nuclear export through the RcRE but that their binding sites are different from each other and from that of Rec.

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

BackgroundMalignant 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).FindingsUsing 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.ConclusionsHERV-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.

Structural organization of unique retrovirus-like particles budding from human teratocarcinoma cell lines.

Human teratocarcinoma cells cultured in vitro can be induced to produce retrovirus-like particles. The induction procedures are the same as those previously shown to induce the synthesis of animal retroviruses. Electron microscopical evidence is presented that the human teratocarcinoma-derived (HTD) particles are most closely related to the type C retrovirus strains. HTD particles can be banded at 1.16 g/ml in linear sucrose gradients, the characteristic density for retroviruses, and subsequently be used for negative staining and fine structure analysis.