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Biomedical subjects

R Kurth

Publications and source records attributed to R Kurth.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1996

Replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus agm3 chimeric viruses in human and monkey cells: the 5' half of the HIV-1 genome is responsible for virus cytopathogenicity.

Two chimeric viruses were constructed between human immunodeficiency virus type 1 (HIV-1) and an apathogenic simian immunodeficiency virus (SIVagm3mc) from African green monkeys. One of the chimeras, HE-A391, expressed the HIV-1-derived env, vpu, tat and rev genes and the SIVagm3mc-derived LTR and the gag, pol and vif genes. The other chimera, SE-H13, contained the SIVagm3mc-derived env, tat and rev genes and the HIV-1-derived LTR and the gag, pol, vif and nef genes. Both constructs yielded infectious viruses and their phenotypes (growth-competence and cell-killing capacity) were examined in various CD4+ cells including human and monkey PBMCs. The results indicated that the replicative properties of the chimeras were mainly dependent on the 5'-genomic half of the parental viruses, and the determinant for viral cytopathogenicity was located within the 5' half of the HIV-1 genome.

Animals↗
PubMed1996

An in vitro assay for acute pathogenicity of immunodeficiency viruses.

As a model for AIDS, experimental infections of old-world monkeys with various simian immunodeficiency viruses (SIV) are frequently carried out to study mechanisms of pathogenicity. For example, SIVsmmPBj14 was isolated from a pig-tailed macaque (Macaca nemestrina) suffering from acute viral disease. The molecular virus clone SIVsmmPBj1.9, which displays close genetic homology to other related SIVs, was shown to induce an acute viral disease in vivo after infection of pig-tailed and rhesus macaques. The acute pathogenicity of SIVsmmPBj1.9 was correlated with its unique ability to replicate in non-stimulated peripheral blood mononuclear cells from pig-tailed macaques. We have exploited this in vitro assay to resolve putative pathogenic genetic determinants of another SIV, namelySIVagm3, isolated from African green monkeys (Cercopithecus aethiops). Hybrid viruses encompassing subgenomic regions of SVsmmPBj1.9 in place of comparable regions of molecular virus clone SIVagm3mc were constructed and tested for their ability to replicate in non-stimulated PBMC from pig-tailed macaques and African green monkeys. Only those hybrid viruses comprising the U3 region of the viral LTR of SIVsmmPBj1.9 replicated in non-stimulated peripheral blood mononuclear cells. This in vitro assay will be used to determine the potential of SIV and of hybrid viruses between different SIVs to induce acute viral disease in vivo. It will help to avoid excessive experimental infections of monkeys with respective hybrid viruses for determining genetic determinants of acute pathogenicity of immunodeficiency viruses.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay.

The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli and eukaryotic enzymes like DNA polymerase alpha and gamma exhibit RT-like activities in this assay, whereas DNA polymerase beta and Klenow enzyme show only minor activities. To discriminate false-positive signals caused by DNA-dependent DNA polymerases in the RT-PCR assay, we have included increasing amounts of activated DNA in the RT reactions. This modification of the assay leads to complete inhibition of aberrant but not authentic RT activities. This improvement specifies the RT-PCR assay as the most sensitive tool for the detection of even very rare replication-competent retroviruses and of related enzymatic activities indigenous, for example, for products of endogenous retrovirus-like RT genes in cell extracts.

Base Sequence↗
PubMed1995

Neither whole inactivated virus immunogen nor passive immunoglobulin transfer protects against SIVagm infection in the African green monkey natural host.

Attempts to protect against infection with simian immunodeficiency virus (SIV)mac grown in rhesus peripheral blood mononuclear cells (PBMCs) using whole inactivated virus immunogen or passive transfer of antibody have so far universally failed. However, such experiments have succeeded in the closely related human immunodeficiency virus (HIV)-2/cynomolgus system. To determine whether the failure in the SIVmac system is typical of primate lentiviruses we performed vaccination and passive transfer experiments using SIVagm (genetically distinct from SIVmac and HIV-2) in the natural African green monkey (AGM) host. To maximize the chances of success, both immunogen and challenge material were prepared using the molecular SIVagm3 clone. Two AGMs were immunized four times with whole inactivated SIVagm3 in RIBI adjuvant and two received intravenously a high dose of immunoglobulin (Ig) purified from a mixture of plasma from AGM, either naturally infected or infected with the homologous SIVagm3 clone. All were challenged with 20 median minimum infective doses of the SIVagm3 grown in AGM PBMCs and previously titrated in AGMs. Despite a strong humoral immune response in all monkeys at the time of challenge, including measurable neutralizing and antibody-dependent cell-mediated cytotoxicity antibodies, none were protected from infection.

Amino Acid Sequence↗
PubMed1995

The U3 promoter region of the acutely lethal simian immunodeficiency virus clone smmPBj1.9 confers related biological activity on the apathogenic clone agm3mc.

Infection with the acutely pathogenic molecular virus clone SIVsmmPBj1.9, cloned from isolate PBj14 of simian immunodeficiency virus (SIV) from sooty mangabey monkeys (Cercocebus atys), leads to acute viral and often lethal disease within days or weeks. SIVsmmPBj1.9 has the unique property of replicating in nonstimulated peripheral blood mononuclear cells from pig-tailed macaques. In contrast, molecular virus clone SIVagm3mc of SIV from African green monkeys (Cercopithecus aethiops), which is apathogenic in its natural host and in pig-tailed macaques, is unable to grow in nonstimulated peripheral blood cells. Chimeric proviruses were constructed by exchanging defined regions of SIVagm3mc against comparable regions of SIVsmmPBj1.9. Four of five hybrid viruses generated by transfection into the CD4-positive T-cell line C8166 replicated in T-cell lines permissive for SIVagm3mc replication and in stimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Three hybrid viruses displayed the distinct biological property of SIVsmmPBj14 to replicate in nonstimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Replication in nonstimulated peripheral blood cells was dependent on the presence of the U3 promoter region of SIVsmmPBj1.9 within the viral long terminal repeat.

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↗
PubMed1995

Rapid screening for Mamu-A1-positive rhesus macaques using a SIVmac Gag peptide-specific cytotoxic T-lymphocyte assay.

As part of an ongoing vaccine study using peptide immunogens designed to stimulate simian immunodeficiency virus (SIV)mac-specific cytotoxic T lymphocytes (CTL) it was necessary to identify rhesus macaques within our colony bearing the Mamu-A1 major histocompatibility complex (MHC) class I haplotype. Peripheral blood mononuclear cells (PBMC) from individual monkeys were analysed by immunoelectrofocusing for the presence of a band corresponding to the Mamu-A1 molecule. In addition, PBMC were pulsed with the SIVmac Gag peptide 11 (against which CTL are Mamu-A1 restricted) and analysed for susceptibility to lysis by peptide 11-specific CTL. PBMC from all of the rhesus macaques shown to be Mamu-A1 positive by immunoelectrofocusing were highly sensitive to lysis by the peptide 11-specific CTL. A total of 46% (16 from 35) of the rhesus macaques originating from India were found to be Mamu-A1 positive, whereas none of the Chinese rhesus (0 from 37) macaques possessed this haplotype. Once a peptide-specific CTL is established, screening by CTL assay offers a faster, reliable and more relevant alternative to immunoelectrofocusing for selecting monkeys for use in vaccination trials.

Amino Acid Sequence↗
PubMed1994

The majority of neutralizing Abs in HIV-1-infected patients recognize linear V3 loop sequences. Studies using HIV-1MN multiple antigenic peptides.

Multiple antigenic peptides (MAP) comprising eight synthetic peptides corresponding to the V3 loop of HIV-1MN or HIV-1IIIB linked to a lysine core were used to characterize the humoral Ab response of HIV-1-infected patients against this domain. One hundred percent of the tested sera from HIV-1-infected European patients reacted significantly with the HIV-1MN V3 loop MAP. Most, but not all, sera also reacted against the HIV-1IIIB V3 loop MAP, albeit with low titers. The in vitro neutralization capacity of the patients' sera against HIV-1MN or HIV-1IIIB infection were tested. All sera showed significant neutralizing titers against HIV-1MN, in comparison with the low (when detectable) neutralizing titers against HIV-1IIIB. Competition assays revealed that the majority of HIV-1MN-neutralizing Abs in patients' sera react with the V3 loop peptides. Only a small fraction of the total neutralizing capacity, presumably specific for other neutralizing epitopes, could not be adsorbed with HIV-1MN V3 loop peptides. The V3 loop peptides could also compete for the binding of Abs in patients' sera mediating Ab-dependent cellular cytotoxicity (ADCC) of HIV-1MN infected cells, inhibiting 30 to 80% of the ADCC activity. Finally, the HIV-1MN V3 loop MAP, when used as immunogen in the presence of RIBI adjuvant without a carrier protein, elicited Abs able to neutralize HIV-1MN (but not HIV-1IIIB) and to initiate ADCC of HIV-1MN-infected cells. This neutralizing and ADCC activity, mediated by V3MN-specific rabbit Abs, could be totally absorbed by V3MN loop peptides.

Amino Acid Sequence↗
PubMed1994

CD8+ T lymphocytes of African green monkeys secrete an immunodeficiency virus-suppressing lymphokine.

Following natural and experimental infection by simian immunodeficiency virus SIVagm of African green monkeys (AGMs), the natural host, there is no evidence for the development of an immunodeficiency. Within the framework of our studies on human immunodeficiency virus (HIV)/SIV pathogenesis, we investigated the influence of CD8 T lymphocytes on SIVagm replication in AGM CD4 T lymphocytes in vitro. The following observations were made: (i) Peripheral blood mononuclear cells from both seronegative and seropositive AGMs contained only a low proportion (i.e., 10%) of CD4+ lymphocytes, whereas a high proportion (80%) of CD8+ cells was detected. Even after persistent SIVagm infection, CD4 T lymphocytes do not decrease in number. (ii) The target of in vitro infection of peripheral blood cells is the CD4+ mononuclear cell (T lymphocytes, monocytes) and SIVagm infects by binding to the CD4 molecule. (iii) In both naturally and experimentally SIVagm-infected AGMs the CD4+ T cells and monocytes, but not the CD8+ T cells, harbor DNA provirus. (iv) Virus reisolation and virus replication of SIVagm in CD4 T lymphocytes from seropositive AGMs is suppressed in the presence of autologous CD8 T lymphocytes or a soluble factor produced by these cells. Taken together, one possible reason for the apathogenicity of the SIVagm infection in AGMs may be the suppression of virus replication by a soluble, yet unidentified factor secreted by CD8 lymphocytes quantitatively dominating among peripheral blood cell populations. We have tentatively termed this factor "immunodeficiency virus-suppressing lymphokine." In addition, we show that immunodeficiency virus-suppressing lymphokine from AGMs is able to suppress HIV-1 replication in human CD4+ T cells.

Animals↗
PubMed1994

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.

3T3 Cells↗
PubMed1994

Rapid inactivation of HIV-1 in single donor preparations of human fresh frozen plasma by methylene blue/light treatment.

Human fresh frozen plasma (FFP) was spiked with highly titered HIV-1 and illuminated with visible light in the presence of 1 microM of the photoactive dye methylene blue (MB). As shown by titration on MT-4 cells, the infectivity of the virus containing plasma was rapidly lost during illumination: after 5 min the infective titer was reduced by 4.3 and after 10 min by at least 6.32 log10, i.e. it was below the detection limit of the assay applied. Methylene blue without illumination and illumination alone had only a marginal effect on HIV-1 infectivity. Thus our data indicate that the MB/light treatment of FFP is an effective method to eliminate the risk of HIV-1 infection through use of the product. This is especially important for those cases in which the plasma is collected during the 'window period' between infection of the donor and the subsequent seroconversion.

Blood↗