Biomedical subjects

M Löchelt

Publications and source records attributed to M Löchelt.

48 records · Page 3Linked to original sources

PubMed1993

Human foamy virus genome possesses an internal, Bel-1-dependent and functional promoter.

The human foamy or spumaretrovirus (HSRV) is a complex retrovirus that encodes the three retroviral genes gag, pol, and env and, in addition, at least three bel genes. The HSRV Bel-1 protein was identified as a transcriptional trans-activator. HSRV transcription starts in the 5' long terminal repeat at a defined guanine residue. We report here that a second efficiently utilized start site of transcription is contained within a HSRV env DNA sequence upstream of the bel genes. Bel-specific transcripts that initiate at the internal transcriptional start site at nt 9196 were identified in HSRV-infected cells by primer extension and S1 nuclease analysis, and the intragenic promoter was shown to be constitutively activated by Bel-1 in the HSRV provirus. In transient expression assays with indicator gene constructs, expression by the HSRV intragenic promoter/enhancer is Bel-1 dependent. The data provide evidence for an intragenic start site of transcription in the genome of a complex, exogenous human retrovirus and are discussed in terms of a model for regulating spumaretroviral gene expression.

Base Sequence↗

PubMed1993

The monomer covalently closed linear replicative form DNA of Aleutian disease parvovirus is infectious after transfection into permissive cells.

The recently described monomer covalently closed linear replicative form DNA (Mccl RF DNA) of Aleutian disease parvovirus (ADV) is an infectious intermediate of the viral DNA replication cycle. Transfection of highly purified Mccl RF DNA into susceptible feline kidney cells (CCC clone 81 cells) resulted in viral DNA replication, expression of viral proteins and synthesis of infectious progeny virus. Mccl RF DNAs generated under permissive (32 degrees C) or non-permissive (37 degrees C) conditions were shown to be biologically indistinguishable. The accumulation of the Mccl RF DNA form at the non-permissive temperature in vitro strongly resembles that in bone marrow cells of naturally infected mink and may reflect one mechanism contributing to virus persistence of ADV in vivo.

Aleutian Mink Disease Virus↗

PubMed1992

Human spumavirus antibodies in sera from African patients.

Serum samples collected from patients with a wide variety of diseases from African and other countries were tested for antibodies to the human spumaretrovirus (HSRV). A spumaviral env-specific ELISA was employed as screening test. Out of 3020 human sera screened, 106 were found to be positive (3.2%). While the majority of patients' sera from Europe (1581) were negative, 26 were positive (1.6%). Sera from healthy adult blood donors (609), from patients with multiple sclerosis (48), Graves' disease (45), and chronic fatigue syndrome (41) were negative or showed a very low prevalence for spumaviral env antibodies. A higher percentage of seropositives (6.3%) were found among 1338 African patients from Tanzania, Kenya, and Gabon. Out of 1180 patients from Tanzania, 708 suffered from tumors, 75 from AIDS, and 128 had gynecological problems; 51 of the Tanzanian patients were HSRV seropositive (4.3%). A particularly high percentage of 16.6% seropositives were identified among nasopharyngeal carcinoma patients (NPC) from Kenya and Tanzania consistent with results reported 10 years ago. However, 20 nasopharyngeal carcinoma patients from Malaysia were HSRV-seronegative. In selected cases, sera from seropositive individuals were reacted with proteins from HSRV-infected cells in vitro. HSRV env- and gag-specific antibodies were specifically detected by these sera in Western blots. The results indicate spumavirus infections in human patients with various diseases at a relatively low prevalence worldwide; in African patients, however, the prevalence of spumavirus infections is markedly higher.

Africa↗

PubMed1991

Construction of an infectious DNA clone of the full-length human spumaretrovirus genome and mutagenesis of the bel 1 gene.

An infectious and full-length molecular clone of genomic human spumaretrovirus (HSRV) DNA was constructed. The infectivity of the pHSRV13 clone was demonstrated after transfection into susceptible cells by passage of HSRV-specific cytopathic effects as a cell-free culture supernatant, by electron microscopy of HSRV particles in pHSRV13 DNA-transfected cells, by detection of HSRV transcripts, and by identification of HSRV-encoded proteins with Env- and Bel-specific antisera in indirect immunofluorescence assays and in protein blotting. The predominant HSRV protein detected in immunoblots by both Bel 1- and Bel 2-specific antisera had an apparent molecular weight of 56 kDa and corresponds to Bet. The amino-terminus of Bet is encoded by part of a Bel 1-specific RNA and the larger Bet domain by an RNA species from the bel 2 gene (Muranyi, W., and Flügel, R. M. J. Virology 65, 727-735, 1991). HSRV-specific proteins of 36 and 43 kDa reacted with Bel 1 and Bel 2 antisera, consistent with the values calculated for the bel 1 and bel 2 gene products, respectively. Deletion mutagenesis of the transcriptional HSRV-specific trans-activator bel 1 and the bet genes completely abolished the infectivity of the pHSRV13 clone. The defect in RNA, protein, and virion synthesis was trans-complemented by cotransfection of an expression clone harboring the complete bel coding region. This result demonstrates that the bel 1 gene is required for viral replication. It remains to be determined whether other HSRV gene products, like bet that share a common region with bel 1, contributed to the defect observed.

Animals↗

PubMed1991

Characterization of the transcriptional trans activator of human foamy retrovirus.

The human foamy viruses, or spumaviruses, a distinct subfamily of complex human retroviruses, remain poorly understood both in terms of their pathogenic potential and in terms of the regulatory mechanisms that govern their replication. Here, we demonstrate that the human spumaretrovirus shares with other complex human retroviruses the property of encoding a transcriptional trans activator of the homologous viral long terminal repeat. This regulatory protein is encoded by the viral Bel-1 open reading frame and is localized to the nucleus of expressing cells. The Bel-1 trans activator is shown to function effectively in cell lines derived from human, simian, murine, and avian sources. The viral target sequence for Bel-1 has been mapped 5' to the start of viral transcription and is therefore likely to be recognized as a DNA sequence. Our results further suggest that the mechanism of action of the Bel-1 protein may be distinct from those reported for the transcriptional trans activators encoded by members of the other human retroviral subfamilies.

Cell Line↗

PubMed1990

Specific enzyme-linked immunosorbent assay for the detection of antibodies to the human spumavirus.

Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV). The corresponding fusion proteins were expressed in E. coli, purified and used subsequently to produce antibodies against the HSRV env proteins in rabbits. The authenticity of the bacterially produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections.

Animals↗

PubMed1990

The monomer covalently closed linear replicative form DNA is an intermediate of Aleutian disease parvovirus DNA replication.

In this report we present data indicating that the recently detected monomer covalently closed linear replicative form DNA (Mccl RF DNA) is an intermediate of Aleutian disease virus (ADV) DNA replication. This DNA molecule is characterized by covalently closed terminal hairpins on either end of the linear ds genomic molecule. Its first detection early after infection in vitro, the association with ADV-specific replication complexes and the de novo synthesis of Mccl RF DNA by isolated replication complexes point to an important role in parvoviral DNA replication. The presence of the Mccl RF DNA in extracts of virus-producing bone marrow cells of naturally infected mink rules out the possibility that this DNA form represents a mere cell culture artefact. The detection of the ADV non-structural protein 1 (NS1) in replication complexes supports the view that this protein is involved in ADV DNA replication.

Aleutian Mink Disease Virus↗

PubMed1990

Mechanisms contributing to the virus persistence in Aleutian disease.

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.

Aleutian Mink Disease↗

PubMed1989

A novel replicative form DNA of Aleutian disease virus: the covalently closed linear DNA of the parvoviruses.

The analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50% of the 5'-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3'-terminal hairpin. This finding together with the presence of nicks in the 3' palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated 'monomer covalently closed linear RF DNA' (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.

Aleutian Mink Disease Virus↗

PubMed1988

Detection of Aleutian disease virus DNA in tissues of naturally infected mink.

Organs of naturally infected mink were examined for the presence of Aleutian disease virus (ADV) DNA by in situ hybridization. Spleen, lymph nodes, thymus, bone marrow, kidney, liver, lung and small intestine were found to be positive for ADV to differing extents. Infected lymphoid organs showed a focal distribution of positive cells. Southern blot analysis of DNA extracted from infected organs revealed replicative forms of viral DNA in spleen and bone marrow samples only. These findings are consistent with a lymphotropism of ADV in vivo. Compared to the situation after experimental infection of mink these results indicate additional sites of virus replication and/or persistence of the naturally occurring disease.

Aleutian Mink Disease↗

PubMed1987

Lymphotropic strain SL3 of Aleutian disease virus: identification of replicative form DNA, molecular cloning and expression of capsid-specific proteins.

Replicative form (RF) DNA of the lymphotropic strain SL3 of Aleutian disease virus was isolated from infected cell cultures. A novel intermediate of about 7.6 kilobases was demonstrated in Hirt lysates in addition to single-stranded viral, double-stranded monomer and dimer RF DNA. The monomer RF DNA exhibited a length heterogeneity of 70 bp and 160 bp at its 3' and 5' termini. The two major monomer RF DNA species each contained hairpins in the extended or the foldback configurations. A central fragment between map units 0.15 and 0.88 was cloned into plasmid pUC18. The recombinant clone expressed virus-specific proteins ranging from 32,000 to 74,000 mol. wt.

Aleutian Mink Disease Virus↗

PubMed1986

Replication of Aleutian disease virus in mink lymphocytes infected in vitro.

The kinetics of Aleutian disease virus (ADV) replication in mink lymphocytes was followed by the analysis of virus-specific antigens, infectious virus, and viral DNA. Stimulation with pokeweed mitogen (PWM; 20 micrograms/ml) increased the synthesis of ADV DNA in cultivated cells of the B-cell fraction. Maximum virus titers [10(6.5) fluorescence-forming units (FFU)/ml] were achieved after incubating infected cells for 60 h at 32 degrees. At this time, 10.4% of the cells in the B-cell-enriched fraction contained ADV-specific antigens and there was an average of 125 ADV genome equivalents per antigen-positive cell. ADV replication also was detected in T-cell-enriched fractions (with up to 10(5.3) FFU/ml, 5.3% of cells were antigen-positive, with 20 ADV genome equivalents per antigen-positive cell), but was 10 times lower compared with ADV replication in the B-cell fraction.

Aleutian Mink Disease Virus↗