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Biomedical subjects

R Löwer

Publications and source records attributed to R Löwer.

At least 19 recordsLinked to original sources
PubMed2004

Detection and characterization of betaretroviral sequences, related to sheep Jaagsiekte virus, in Africans from Nigeria and Cameroon.

Jaagsiekte retrovirus (JSRV) causes ovine pulmonary adenomatosis (OPA) that resembles bronchioloalveolar carcinoma (BAC) in humans. To test the possible role of JSRV in human diseases, DNA specimens from 103 individuals either healthy or suffering from lung carcinomas were analyzed for JSRV sequences. orf-x sequences were detected in 19 of 64 samples and gag-prt sequences in 4 of 38 samples, predominantly in individuals from Africa. Sequences obtained from orf-x amplimers varied in-between each other and differed from control endogenous ovine JSRV sequence. No association with lung cancer was found. This is the first report of JSRV-like sequences detected in humans.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2004

Conservation and loss of the ERV3 open reading frame in primates.

The human endogenous retrovirus ERV3 possesses an open reading frame for a truncated envelope, which is expressed as mRNA and protein. Here we examine the env sequence in primates for evidence of evolutionary conservation. ERV3 sequences were amplified by PCR from genomic DNA of great ape and Old World primates but not from New World primates or gorilla, suggesting an integration event more than 30 million years ago with a subsequent loss in one species. In the chimpanzee, the protein sequence of Env is 98.18% identical to that of human. In other species the identity falls (93.71% in rhesus macaque) in proportion to the separation from the human lineage. Start and stop codons and domains of functional significance in the envelope protein are conserved. The evolutionary conservation of the ERV3 envelope suggests a beneficial function, though the loss from gorilla shows that it is not essential for survival or reproduction.

Amino Acid Sequence↗
PubMed2001

Rapid evaluation and optimization of recombinant protein production using GFP tagging.

The isolation of recombinant proteins from bacterial or eukaryotic systems often requires a laborious optimization of expression and purification conditions. To greatly facilitate this procedure we included the green fluorescent protein (GFP) in bacterial expression vectors. This approach allowed us to sensitively detect the GFP hybrid proteins already in intact bacterial cells using a fluorescence microscope. To rapidly analyze a variety of conditions essential for protein expression, the GFP signal, indicative of expression levels, was directly quantitated in live bacterial suspensions using a fluorescence plate reader. Thus, GFP tagging not only allows one to directly monitor protein expression in general but also appears to increase protein stability or solubility.

Cloning, Molecular↗
PubMed2001

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.

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.

Base Sequence↗
PubMed2000

[Eosinophilic myocarditis in Churg-Strauss syndrome. A rare cause of left heart decompensation with lung edema].

HISTORY: A 50-year-old woman was admitted because of marked dyspnoea at rest and signs of left heart failure with pulmonary oedema. 9 years ago, the diagnostic constellation of bronchial asthma, polyneuropathy, pericardial effusion and eosinophilia had indicated Churg-Strauss syndrome. Since then she had remained symptom-free under maintenance doses of azathioprine (for 2 years) and gradually reduced doses of steroids. INVESTIGATIONS: Chest X-ray showed signs of pulmonary congestion and cardiomegaly, echocardiography demonstrating enlargement of the left heart with marked impairment of ventricular function, and both revealed pericardial effusion. The electrocardiogram showed complete absence of R waves and ST elevation in leads V1-V5. Coronary angiography excluded coronary artery disease. Myocardial biopsy contained signs of active but no longer acute myocarditis with eosinophilic tissue infiltration and microgranulomas. White blood cell count was normal, but there was marked eosinophilia (39%). IgE was elevated (601 kIU/l). DIAGNOSIS, TREATMENT AND COURSE: In view of the good therapeutic effects 9 years ago, this relapse of Churg-Strauss syndrome with eosinophilic myocarditis was again treated with azathioprine and steroids. In addition, diuretics, digitalis and ACE-inhibitors successfully treated the heart failure. In the course of treatment the signs of inflammation, including the eosinophilia, regressed or became normal. CONCLUSION: After a 10-year remission without complication of a Churg-Strauss syndrome the onset of cardiac signs is the decisive long-term prognostic factor.

Biopsy↗
PubMed2000

Corf, the Rev/Rex homologue of HTDV/HERV-K, encodes an arginine-rich nuclear localization signal that exerts a trans-dominant phenotype when mutated.

Regulation of nucleo-cytoplasmic export of viral transcripts by a viral protein (Rev/Rex) is a characteristic feature in the replication cycle of complex retroviruses. We recently reported that the endogenous retrovirus family HTDV/HERV-K encodes a protein, Corf, that is a cellular Counterpart of Rev/Rex function and thus a new component of nucleo-cytoplasmic pathways. In HTDV/HERV-K-expressing cells, Corf is localized within the nucleoli. Here we describe the nuclear localization signal (NLS) of the Corf protein. Mutations in the NLS lead to cytoplasmic accumulation of the mutated protein and abrogate Corf function in a trans-dominant way.

Amino Acid Sequence↗
PubMed2000

Functional analysis of the env open reading frame in human endogenous retrovirus IDDMK(1,2)22 encoding superantigen activity.

Mice harbor a family of endogenous retroviruses, the mouse mammary tumor viruses (MMTV), which encode superantigens. These superantigens are responsible for the deletion of T cells expressing certain Vbeta chains of the T-cell receptor in the thymus. Human T cells are able to recognize MMTV-encoded superantigens presented by human major histocompatibility complex class II-positive cells. Owing to this and to the similarity of the human and murine immune systems, it was speculated that human endogenous retroviruses might also code for superantigens. Recently, it was reported that a proviral clone (IDDMK(1,2)22) of the human endogenous retrovirus family HTDV/HERV-K encodes a superantigen. The putative superantigen gene was located within the env region of the virus. Stimulated by these findings, we amplified by PCR and cloned into eucaryotic expression vectors open reading frames (ORFs) which were identical or very similar to IDDMK(1,2)22. When we transfected these vectors into A20 cells, a murine B-cell lymphoma, we were able to demonstrate mRNA expression and protein production. However, we did not find any evidence that the ORF stimulated human or murine T cells in a Vbeta-specific fashion, the most prominent feature of superantigens.

Animals↗
PubMed1999

The pathogenic potential of endogenous retroviruses: facts and fantasies.

Endogenous retroviruses are descendants of viruses that became cellular genes by integration into their host's 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.

Animals↗
PubMed1999

DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas.

Since DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome, hypomethylation in human tumours may lead to their reactivation. The methylation status of LINE-1 retroposons was determined in 73 samples of urinary bladder cancers, 34 specimens of renal cell carcinoma and in the corresponding normal tissues by Southern blot analysis. LINE-1 sequences were strongly methylated in normal tissues and were significantly hypomethylated in 69 (95%) urothelial carcinomas, but in none of the renal carcinomas. Hypomethylation in bladder cancers was independent of stage and tended to increase with grade. The methylation status of HERV-K proviral DNA in normal and transformed urothelial cells paralleled that of LINE-1 sequences (r2 = 0.87). It was shown by ligation-mediated polymerase chain reaction that hypomethylation also extended to the LINE-1 promoter sequence located at the 5'-ends of full-length elements which is repressed by methylation in somatic tissues. Accordingly, full-length LINE-1 transcripts were detected by Northern blot analysis in two urothelial carcinoma cell lines. In contrast, transcripts from HERV-K proviruses were restricted to teratocarcinoma cell lines. Our data indicate that genome-wide DNA hypomethylation is an early change in urothelial carcinoma, but is absent from renal cell carcinoma. The coordinate changes of LINE-1 and HERV-K DNA methylation suggest that hypomethylation in urothelial cancer affects a variety of different retroelements to similar extents. We speculate that decreased methylation of LINE-1 retroelements, in particular, may contribute to genomic instability in specific human tumours such as urothelial carcinoma by rendering these normally repressed sequences competent for transcription and recombination.

Adult↗
PubMed1999

cORF and RcRE, the Rev/Rex and RRE/RxRE homologues of the human endogenous retrovirus family HTDV/HERV-K.

cORF, a protein encoded by the human endogenous retrovirus family HTDV/HERV-K, contains amino acid motifs which resemble the nuclear import and export signals of the viral regulatory proteins Rev (human immunodeficiency virus) and Rex (human T-cell leukemia virus [HTLV]). In this study, we demonstrated that cORF indeed has a Rev-like function and mapped the cORF-responsive RNA element to a sequence in the 3' long terminal repeat, a localization similar to RxRE, the responsive element in HTLV type 1. Accordingly, we have given the element the designation RcRE. cORF and RcRE stabilize unspliced and incompletely spliced viral transcripts and enhance their nuclear export via the CRM1 export pathway. So far, HTDV/HERV-K is the only endogenous retrovirus family with a complex regulation at the posttranscriptional level. It may be regarded as an intermediate in the evolution from simple to complex retroviruses.

Amino Acid Sequence↗
PubMed1999

Expression of the human endogenous retrovirus HTDV/HERV-K is enhanced by cellular transcription factor YY1.

The human endogenous retrovirus HTDV/HERV-K, which resides in moderate copy numbers in the human genome, is expressed in a cell-type-specific manner, predominantly in teratocarcinoma cells. We have analyzed the regulatory potential of the 5' enhancer of the HERV-K long terminal repeat. Protein extracts of HERV-K-expressing teratocarcinoma cell lines (GH and Tera2) and nonexpressing HeLa and HepG2 cells form different protein complexes on the enhancer sequence as detected by electrophoretic mobility shift assays (EMSA). Using competition EMSAs, DNase I footprinting, and supershift experiments, we localized the binding site of these complexes to a 20-bp sequence within the enhancer and showed that the transcription factor YY1 is one component of the HERV-K enhancer complex. Replacement of the YY1 binding site with unrelated sequences reduced expression of the luciferase gene as a reporter in transient-transfection assays.

DNA-Binding Proteins↗
PubMed1997

Expression of human endogenous retrovirus type K envelope glycoprotein in insect and mammalian cells.

The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles.

Animals↗
PubMed1996

Endogenous D-type (HERV-K) related sequences are packaged into retroviral particles in the placenta and possess open reading frames for reverse transcriptase.

All primates studied to date produce retroviral-like particles in their placentae. We have purified these particles from two primate species, one Old World (human) and one New World (marmoset), and have identified the retroviral sequences which are packaged into these particles. Three families of sequences have been detected in these particles in human, all of which have the highest homology to B- and D-type retroviruses and to the human endogenous retrovirus HERV-K10. Previous studies have reported that the New World monkeys do not possess sequences with homology to HERV-K10. We have identified a new family of low-copy-number sequences which are present in New World monkeys and which possess 70% homology to the HERV-K family. Particles from both species possess reverse transcriptase activity and we have found that some of these retroviral particles package sequences which encode long open reading frames in pol, as revealed by expression cloning in Escherichia coli. These open reading frames could encode the reverse transcriptase enzyme activity found in the particles.

Animals↗
PubMed1996

The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.

Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.

Antibody Formation↗
PubMed1996

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.

Animals↗
PubMed1995

Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K.

The human endogenous retrovirus family HTDV/HERV-K codes for the viral particles observed in teratocarcinoma cell lines. Two types of proviral genomes exist; these differ in the presence or absence of a stretch of 292 nucleotides. This sequence comprises the amino-terminal part of the env gene, the putative signal peptide, which overlaps in part with the carboxy terminus of the pol gene. Type 2 genomes containing this sequence presumably more closely reflect the structure of the infectious, replication-competent retrovirus ancestors of the HERV-K family than do type 1 genomes that lack the sequence. In human teratocarcinoma cell lines, both variants are expressed. Type 1 genomes, in which pol and env genes are fused, are deficient in splicing. Type 2 transcripts are spliced to subgenomic env mRNA and smaller messages. A doubly spliced transcript encodes a short open reading frame, preliminarily designated cORF (R. Löwer, K. Boller, B. Hasenmeier, C. Korbmacher, N. Mueller-Lantzsch, J. Löwer, and R. Kurth, Proc. Natl. Acad. Sci. USA 90:4480-4484). The genomic organization of cORF resembles that of nonprimate lentivirus rev genes: the first exon comprises nearly the entire signal peptide of env, and the second exon is derived from a different reading frame in the 3' part of the genome. A nucleolar localization signal, which is also a putative RNA binding domain, as well as a sequence with similarities to the Rev effector domain consensus sequence is present in the first exon. Secondary structure analysis reveals similarities to basic helix-loop-helix proteins. cORF is a small protein with an apparent molecular mass of 14 kDa which accumulates in the nucleolus as has been described for Rev proteins.

Amino Acid Sequence↗
PubMed1993

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

Base Sequence↗