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V Erfle

Publications and source records attributed to V Erfle.

At least 55 records · Page 3Linked to original sources
PubMed1996

Biological significance of human endogenous retroviral sequences.

Endogenous retroviruses (ERVs) have been known for many years to exist in numerous natural and laboratory animal species. In humans it has been demonstrated that at least 1% of the genome consists of retrovirus-related sequences. Involvement of ERVs in the development of neoplastic and autoimmune diseases in the mouse model implicated a potentially pathogenic role of ERVs for humans, too. The research in this field led to a number of results strongly suggesting that human endogenous retroviral sequences (HERVs) are biologically active, on the RNA and even on the protein level. Particle formation, regulation or dysregulation of cellular gene expression, and synthesis of potentially pathogenic viral proteins indicate the broad spectrum of mechanisms by which HERVs may obtain biological significance.

Animals↗
PubMed1996

A new variant of B1 elements, vB1, identified upstream of a retroviral integration site.

A new variant of the murine B1 retroposon family, vB1, was identified in antisense orientation 165 bases upstream of the integration site of RFB MuLV in infected NIH3T3 fibroblasts. vB1 revealed a characteristic B1 structure, but contained two additional unique repeats. This variant facilitates identification of related vB1 elements which are considered to represent a minor subgroup of B1 elements.

3T3 Cells↗
PubMed1995

Characterization of a protein that binds a negative regulatory element in the mammary-specific whey acidic protein promoter.

Whey Acidic Protein (WAP) gene expression is restricted to the pregnant and lactating mammary gland. We have recently defined a negative regulatory element (NRE) in the WAP promoter which interacts with a factor (NBF) present in all nonWAP expressing cells (Kolb et al., 1994; J. Cell. Biochem. 56:245-261). Here we characterise this factor and show that although it is not related to a number of known transcription factors, including AP-1, NF-1 and SP-1, it may also be involved in controlling the expression from the mouse mammary tumour virus promoter. Three proteins that bind to the WAP-NRE have been identified, one of which is a 53kDa nuclear protein. This protein is present in nonWAP expressing cells, suggesting that it is responsible for limiting WAP expression to the pregnant and lactating mammary gland. This protein has been partially purified and its binding to the WAP-NRE is not appreciably affected by high salt concentrations.

Animals↗
PubMed1995

Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase.

Modified vaccinia virus Ankara (MVA), a host range restricted and highly attenuated vaccinia virus strain, is unable to multiply in human and most other mammalian cell lines. Since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses are efficient as well as exceptionally safe expression vectors. We constructed a recombinant MVA that expresses the bacteriophage T7 RNA polymerase and tested its usefulness for transient expression of recombinant genes under the control of a T7 promoter. Using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene, infection with MVA-T7pol allowed efficient synthesis of recombinant enzyme in mammalian cells. Despite the severe host restriction of MVA, enzyme activities induced by infection with MVA-T7pol were similar to those determined after infection with a replication-competent vaccinia-T7pol recombinant virus. Thus, MVA-T7pol may be used as a novel vaccinia vector to achieve T7 RNA polymerase-specific recombinant gene expression in the absence of productive vaccinia virus replication.

Animals↗
PubMed1995

Identification of S71-related human endogenous retroviral sequences with full-length pol genes.

The human genome contains sequences related to the simian sarcoma-associated virus SSAV. One of these endogenous retroviral elements, S71, is truncated in the pol gene and carries an insertion of a solitary HERV-K LTR. Using a PCR approach we have now identified further S71-related retroviral elements that lack the HERV-K LTR insertion and contain a full-length retroviral reverse transcriptase. Two of these sequences, pCRTK1 and pCRTK6, were cloned and further characterized. Clones pCRTK1 and pCRTK6 showed between 85 and 90% nucleotide homology to each other and to S71 within the "tether" region of the pol gene, indicating that pCRTK1 and pCRTK6 clearly belong to the S71 subgroup of C-type-related human endogenous retroviral elements. Some point mutations inactivating the reverse transcriptase are located at the same positions in pCRTK1 and pCRTK6. Therefore, we assume that these S71-related elements were dispersed in the human genome by reintegration as defective proviruses, probably using enzymes for retrotransposition provided in trans by other retrotransposons or by cellular genes. Examination of the presence of S71-related elements in apes and Old World monkeys revealed that the deletion of reverse transcriptase sequences in S71 has occurred in the lineage of primates prior to the insertion of the HERV-K LTR.

Amino Acid Sequence↗
PubMed1995

Negative-acting factor and superantigen are separable activities of the mouse mammary tumor virus long terminal repeat.

The open reading frame contained within the long terminal repeat (LTR) of mouse mammary tumor virus encodes Naf, a negative regulator of transcription, as well as a superantigen activity, Sag, which causes the deletion of specific classes of T cells. In the present study, the effect of Naf expression on different promoters and the coding requirements for Naf and Sag have been investigated. Sag activity was found to require only sequences in the LTR, whereas sequences located within the gag gene were additionally required for functional Naf activity. Surprisingly, both the classic promoter and a recently described promoter located in the LTR can give rise to both functional Naf and Sag. Further analysis of Naf revealed that the downregulatory effect was mediated by sequences located in the LTR and that heterologous promoters were also affected by Naf.

Animals↗
PubMed1995

Akv murine leukemia virus enhances bone tumorigenesis in hMT-c-fos-LTR transgenic mice.

hMt-c-fos-LTR transgenic mice (U. Rüther, D. Komitowski, F. R. Schubert, and E. F. Wagner. Oncogene 4, 861-865, 1989) developed bone sarcomas in 20% (3/15) of females at 448 +/- 25 days and in 8% (1/12) of males at 523 days. After infection of newborns with Akv, an infectious retrovirus derived from the ecotropic provirus of the AKR mouse, 69% (20/28) of female animals and 83% (24/29) of males developed malignant fibrous-osseous tumors. The tumors in infected transgenics developed with higher frequency and a 200-days shorter mean tumor latency period. The hMt-c-fos-LTR transgene was expressed in all the fibrous-osseous tumors. They also showed newly integrated Akv proviruses, but in most tumors Akv was detected and expressed in only a small number of the tumor cells. Wild-type C3H mice infected with Akv developed benign osteomas with an incidence of 33% and a latency period of 474 days. The data indicate that Akv exerts distinct pathogenic effects on the skeleton. In hMt-c-fos-LTR transgenic mice, predisposed to bone sarcomagenesis, Akv acts synergistically with the fos transgene, resulting in the development of fibrous-osseous tumors.

AKR murine leukemia virus↗
PubMed1995

Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice.

The contribution of endogenous retroviruses to the multistep process of lymphomagenesis was investigated in wild-type mice and in two different myc-kappa transgenic mouse lines by infection with Akv. This retrovirus is derived from the endogenous ecotropic provirus of the AKR mouse and was previously considered to be nonlymphomagenic. The mice of the two myc-k transgenic lines are predisposed to B-cell lymphomagenesis and were therefore considered to be more susceptible to Akv. For comparison, the same mouse strains were also infected with the exogenous Moloney murine leukemia virus (MoMuLV). Both MoMuLV and Akv increased the tumor incidence and shortened the tumor latency period in wild-type mice and in the transgenic mouse lines. The differences in pathogenicity, number of provirus integrations, and level of virus expression between MoMuLV and Akv indicate different mechanisms of lymphomagenesis: while MoMuLV induced tumors apparently by insertional mutagenesis involving common integration sites similar to previous reports, the enhancement of lymphomagenesis by Akv seems to be directed by other mechanisms.

AKR murine leukemia virus↗
PubMed1995

Cleavage of recombinant and cell derived human immunodeficiency virus 1 (HIV-1) Nef protein by HIV-1 protease.

Recombinant purified Nef protein of HIV-1, as well as Nef protein derived from extracts of permanently HIV-1 infected glioblastoma cells and monocytes, are specifically cleaved by the HIV-1 protease. Nef cleavage products in cellular extracts treated with protease showed identical molecular weights as those obtained by digestion of purified Nef with recombinant HIV-1 protease. Since cellular extracts were prepared by detergent and mechanical lysis it cannot be excluded that physiological cytoplasmic conditions were altered. The lack of Nef cleavage by endogenous HIV-1 protease in infected cells might be due to low concentrations of viral protease and the presence of Gag precursor molecules as natural substrate. Using a panel of monoclonal antibodies two cleavage fragments of 19 kDa and 8 kDa were defined. The cleavage site was located by microsequencing between amino acid 57 and 58 (AW*LEAQEEEEVGF). The conserved cleavage motif within HIV-1 Nef suggests a potential biological function of Nef processing.

Amino Acid Sequence↗
PubMed1995

Identification of three human sequences with viral superantigen-specific primers.

The open reading frame (ORF) in the long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) has recently been shown to encode multiple products including a negative acting factor (Naf) and a superantigen (Sag). Expression of superantigens from endogenous MMTV loci in the mouse results in the deletion of whole classes of T cells. In a PCR approach, with primers to the MMTV ORF and hybridization to MMTV specific probes, we have identified three human sequences. Direct sequencing of PCR products revealed that one of these products is related to a human autoantigen that is conserved among many species and is expressed in testes and sperm. The second sequence that we have identified is novel, and no evidence for expression of this sequence could be obtained. Finally, the third ORF-like sequence is a new member of a previously described family of human endogenous retroviruses (RTVL-I). This sequence is transcribed in several human cell lines, including B lymphoblastoid cells, and is thus the first demonstration that an RTVL-I-related sequence can be expressed. Taken together, these findings raise the intriguing possibility that the human genome contains superantigen-like sequences, some of which are also related to endogenous retroviruses, that may influence the T cell repertoire.

Amino Acid Sequence↗
PubMed1995

Distribution of HIV genomic DNA in brains of AIDS patients.

BACKGROUND: Data concerning the distribution of HIV in the brains of AIDS patients at different stages of viral infection might contribute towards: (1) understanding the route(s) of HIV entry into the brain and virus dissemination within the brain and (2) establishing a possible correlation between the extent of CNS damage and the distribution of virus in AIDS brains. OBJECTIVE: To determine the distribution of HIV-1 genomic DNA within the brains of three deceased AIDS patients by polymerase chain reaction (PCR). STUDY DESIGN: The brains of three deceased AIDS patients were examined. Two brains had limited neuropathologic findings (brains I and II), and one brain (brain III) showed primary HIV-specific neuropathologic damage. Tissues were taken from different locations within each brain, and high molecular weight DNA isolated from the tissues was assessed for HIV-1 genomic DNA by PCR. RESULTS: HIV-1 genomic DNA was found in all three brains, but the amount was low: order of magnitude of 1 viral genome per 1,000 cells. Multiple PCR analyses of DNA from brain I showed that the viral genomic DNA in this brain was non-uniformly distributed; only samples taken from the brainstem were clearly positive for HIV-1. HIV-1 genomic DNA in brain II was found in portions of the lower and upper hemispheres. All but one of the brain III samples were clearly positive for HIV-1, and they had been taken from locations spread throughout this brain. CONCLUSIONS: Our results suggest that in early or latent stages of HIV-infection of the brain, viral genomic DNA is localized at restricted regions. At later stages this DNA is distributed more uniformly throughout the brain. Our data are compatible with the concept of rare infection events followed by viral spreading within brain tissues.

Journal Article↗
PubMed1995

Restriction of human immunodeficiency virus type 1 production in a human astrocytoma cell line is associated with a cellular block in Rev function.

Chronically human immunodeficiency virus type 1 (HIV-1) strain IIIB-infected human TH4-7-5 astrocytoma cells show low-level virus production. Cocultivation of TH4-7-5 cells with myelomonocytic cells led to active virus production in these target cells after a lag period, indicating cell-determined restriction of virus replication in the glial cells. HIV-1 transcript patterns of TH4-7-5 cells contained only a small proportion of Rev-dependent mRNA species, mimicking a Rev-negative phenotype despite the presence of rev mRNAs and protein. Sequencing of the single provirus integrated in TH4-7-5 cells demonstrated that the rev gene and the Rev-responsive element are intact. These results suggested inhibited function of the Rev-regulatory unit in these astrocytoma cells. Transfection of TH4-7-5 cells with a Rev expression plasmid resulted in weak or no induction of proviral p24gag antigen levels compared with the dramatic increase observed in Rev-permissive HeLa cells. Immunofluorescence analysis of TH4-7-5 cells transfected with a rev-expressing plasmid revealed prominent cytoplasmic and nuclear-nucleolar localization of Rev, in contrast to the predominant nuclear-nucleolar localization pattern of Rev in HeLa cells. We conclude that restriction of virus production in TH4-7-5 cells is at least partially due to a block in Rev-dependent posttranscriptional regulation of HIV expression.

Amino Acid Sequence↗
PubMed1995

Genetic instability of a dinucleotide repeat-rich region in three hematologic malignancies.

Microsatellite instability is a newly identified mechanism of mutation that occurs in some heritable neurological and muscular disorders, as well as in an increasing number of human cancers. To extend previous data, we examined the genetic instability of a human genomic region, termed S3/1, which we isolated from a human DNA library. The S3/1 sequence contains a stretch with exceptionally high numbers of (GA)n and (CA)n dinucleotide repeats. An interesting rearranged pattern emerged from Southern blot analysis of genomic DNA from three patients with different hematopoietic proliferative diseases out of 69 analyzed (one case of essential thrombocytosis (ET), one of chronic myelogenous leukemia (CML) and one of acute myelogenous leukemia (AML)). The CML and ET patients showed a deletion of 300 to 400 base pairs (bp), and the AML an insertion of about 600 bp, involving the S3/1 locus. Amplification of the rearranged fragments confirmed these observations, and enabled a precise analysis of the region involved. In normal individuals, no gross rearrangements involving this region could be detected. Analysis of DNA from three consecutive bone marrow biopsies of the CML patient disclosed that the genetic alteration affecting S3/1 was no longer detectable following alpha 2-interferon therapy, neither by Southern blot nor by polymerase chain reaction (PCR), thus confirming the tumor-specificity of the alteration; in the same patient, moreover, two out of five other analyzed microsatellites showed tumor-specific alleles, suggesting a more generalized genetic instability in the leukemic cells. These results demonstrate genetic instability of a region containing high numbers of short dinucleotide repeats in a small percentage (4%) of human hematopoietic proliferative disorders.

Acute Disease↗
PubMed1994

Disruption of the murine p53 gene by insertion of an endogenous retrovirus-like element (ETn) in a cell line from radiation-induced osteosarcoma.

The mammalian genome harbors a large number of endogenous retroviruses and retrovirus-like elements. Increasing evidence is found that such elements can be activated and act as insertional mutagens. The activation of endogenous retroviral elements can be induced by a variety of environmental factors including irradiation. We have observed the insertion of a murine endogenous retrovirus-like ETn element into intro 4 of the p53 gene in an osteosarcoma cell line derived from a radiation-induced osteosarcoma. The insertion resulted in a p53-ETn-p53 fusion mRNA, a novel form of p53 mutation. This is the first report on insertion of an endogenous retroviral element into the p53 tumor suppressor gene. The data suggest that activated endogenous retroviruses and retrovirus-like elements might pose an enhanced risk for individuals exposed to noxae, which activate endogenous retroviral elements.

Animals↗
PubMed1994

Negative regulatory element in the mammary specific whey acidic protein promoter.

Expression of the whey acidic protein (WAP) gene is tightly regulated in a tissue and developmental stage specific manner, in that the WAP gene is exclusively expressed in the mammary gland during pregnancy and lactation. Using both deletion and competition analyses, evidence is provided for the existence of a negative regulatory element (NRE) in the WAP promoter located between -413 and -93 with respect to the WAP transcriptional initiation site. This NRE dramatically decreases transcription from linked heterologous promoter-reporter gene constructs. The activity of NRE requires WAP promoter sequences that are 230 bp apart since subfragments of the NRE fail to inhibit transcription of adjoining reporter genes. Nuclear extracts from different cell types, in which the WAP gene is not active, contain a protein or complex that specifically interacts with the entire NRE but not with subfragments of it. The contact points between this protein (NRE binding factor [NBF]) and the NRE element have been partially determined. Mutation of the implicated nucleotides severely reduces the ability of NBF to bind, and such mutated promoter fragments fail to alleviate transcriptional repression in competition experiments. This suggests that NBF binding to the NRE is at least in part responsible for the negative regulation of the WAP promoter. Since NBF is not detectable in the lactating mammary gland, where the WAP gene is expressed, we speculate that it may be a determinant of the expression spectrum of the WAP gene.

Animals↗
PubMed1994

Osteosarcomagenic doses of radium (224Ra) and infectious endogenous retroviruses enhance proliferation and osteogenic differentiation of skeletal tissue differentiating in vitro.

Cartilage tissue from embryonic mice which undergoes osteogenic differentiation during in vitro cultivation was used to study the effect of osteosarcomagenic doses of alpha-irradiation and bone-tumor-inducing retroviruses on proliferation and phenotypic differentiation of skeletal cells in a defined tissue culture model. Irradiated mandibular condyles showed dose-dependent enhancement of cell proliferation at day 7 of the culture and increased osteogenic differentiation at day 14. Maximal effects were found with 7.4 Bq/ml of 224Ra-labeled medium. Doses of 740 and 7400 Bq/ml of 224Ra-labeled medium induced increasing cell death. Retrovirus infection enhanced osteogenic differentiation and extended the viability of irradiated cells. After transplantation none of the treated tissues developed tumors in syngeneic mice.

Animals↗
PubMed1994

Adipose differentiation of cartilage in vitro.

Xiphoids of newborn mice consist of young chondrogenic cells of primary cartilage. During in vitro cultivation, xiphoids showed, morphologically, characteristics of adipose differentiation. This process progressed with time and by day 21 of the culture most of the cells in the xiphoids represented morphological mature adipocytes. During this period, the level of mRNA of lipoprotein lipase, and adipocyte-characteristic gene, increased steadily, while the level of collagen type II mRNA decreased. Continuous DNA synthesis during the cultivation period, even in mature adipocytes confirmed the viability of the cells. Mandibular condyles of newborn mice obtain chondroprogenitor cells as well as young and mature chondroblasts and represent secondary cartilage. Under identical culture conditions mandibular condyles obtained from the same mice undergo osteogenic differentiation and form mature bone within 7 to 10 days. Common to both xiphoids and mandibular condyles is the capacity to transdifferentiate, but they show distinct, divergent differentiation pathways. These findings indicate that cartilagenous tissue of xiphoids undergoes transdifferentiation into adipose tissue in vitro.

Adipose Tissue↗