Biomedical subjects
P Staeheli
Publications and source records attributed to P Staeheli.
cDNA cloning of biologically active chicken interleukin-18.
By searching a chicken EST database, we identified a cDNA clone that appeared to contain the entire open reading frame (ORF) of chicken interleukin-18 (ChIL-18). The encoded protein consists of 198 amino acids and exhibits approximately 30% sequence identity to IL-18 of humans and various others mammals. Sequence comparisons reveals a putative caspase-1 cleavage site at aspartic acid 29 of the primary translation product, indicating that mature ChIL-18 might consist of 169 amino acids. Bacterially expressed ChIL-18 in which the N-terminal 29 amino acids of the putative precursor molecule were replaced by a histidine tag induced the synthesis of interferon-gamma (IFN-gamma) in cultured primary chicken spleen cells, indicating that the recombinant protein is biologically active.
Isolation and characterization of a new subtype of Borna disease virus.
Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus Bornavirus of the family Bornaviridae. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.
Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells.
The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.
Chemokine gene expression in astrocytes of Borna disease virus-infected rats and mice in the absence of inflammation.
Borna disease virus (BDV) causes CD8(+) T-cell-mediated meningoencephalitis in immunocompetent mice and rats, thus providing a valuable animal model for studying the mechanisms of virus-induced central nervous system (CNS) immunopathology. Chemokine-mediated leukocyte recruitment to the CNS is a crucial step in the development of neurological disease. We found increased mRNA levels of IP-10 and other chemokines in brains of adult rats following infection with BDV. The marked increase in chemokine gene expression at about day 8 postinfection seemed to immediately precede the inflammatory process. In brains of rats infected as newborns, in which inflammation was only mild and transient, sustained expression of IP-10 and RANTES genes was observed. In situ hybridization studies revealed that astrocytes were the major source of IP-10 mRNAs in brains of rats infected as newborns and as adults. In brains of infected mice lacking CD8(+) T cells (beta2m(0/0)), transcripts encoding IP-10 and RANTES were also observed. IP-10 transcripts were also present in a small number of scattered astrocytes of infected knockout mice lacking mature B and T cells as well as functional alpha/beta and gamma interferon receptors, indicating that BDV can induce chemokine synthesis in the absence of interferons and other B- or T-cell-derived cytokines. These data provide strong evidence that CNS-resident cells are involved in the early localized host immune response to infection with BDV and support the concept that chemokines are pivotal for the initiation of virus-induced CNS inflammation.
Chromosomal localization of the genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma, and VIM in chicken by fluorescence in situ hybridization.
Six structural genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma and VIM were mapped in the chicken by fluorescence in situ hybridization (FISH) using genomic and cDNA clones as probes. The genes were found to be located on four different macrochromosomes: chromosome 1 (IFNG and FABP), chromosome 2 (VIM and ALDH), chromosome 3 (BMP2) and a smaller macrochromosome, most probably chromosome 7 (RXRG). With the exception of IFNG none of the newly mapped sites corresponds to known orthologous regions between chicken and human chromosomes.
Sequence similarities between human bornavirus isolates and laboratory strains question human origin.
Human bornavirus RW98, whose genome differs by 3-4% from previous isolates, is almost identical to a rat-adapted laboratory strain. Other human bornaviruses are also strongly related to virus strains frequently used for experiments in the various laboratories reporting human bornavirus, questioning a human origin of isolates known to date.
T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein.
Infection of neonates with Borna disease virus (BDV) induces severe meningoencephalitis and neurological disorder in wild-type but not in beta(2)-microglobulin-deficient mice of strain MRL (H-2(k)). Temporary in vivo depletion of CD8(+) T cells delayed BDV-induced disease for several weeks. Depletion of CD4(+) T cells had a similar beneficial effect, indicating that the BDV-induced neurological disorder in mice is a CD4(+) T cell-dependent immunopathological process that is mediated by CD8(+) T cells. Lymphocytes prepared from brains of diseased mice were mainly from the CD8(+) T cell subset. They showed up-regulation of activation markers and exerted strong MHC I-restricted cytotoxic activity against target cells expressing the BDV nucleoprotein p40. Infection of B10.BR (H-2(k)) or congenic C57BL/10 (H-2(b)) mice resulted in symptomless, lifelong persistence of BDV in the brain. Superinfection with a recombinant vaccinia virus expressing BDV p40 but not with other vaccinia viruses induced severe neurological disease and encephalitis in persistently infected B10.BR mice but not in persistently infected C57BL/10 mice, indicating that the disease-inducing T cell response is restricted to the nucleoprotein of BDV in H-2(k) mice. Our results demonstrate that the cellular arm of the immune system may ignore the presence of a replicating virus in the central nervous system until proper antigenic stimulation at a peripheral site triggers the antiviral response.
Protective effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens.
Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.
Inhibition of Borna disease virus multiplication by interferon: cell line differences in susceptibility.
It has previously been reported that de novo infection of primary rabbit brain cells with Borna disease virus (BDV) can be blocked with interferon-alpha/beta (IFN), whereas this cytokine has no inhibitory effect on BDV in persistently infected rat lung cells [v. Rheinbaben et al., J. Gen. Virol. (1985) 66: 2,777-2,780]. It remained unclear, however, whether these results indicated that IFN exclusively targets early steps of the BDV replication cycle or whether they simply reflected cell line differences. We now show that BDV replication was effectively inhibited by IFN in both acutely and persistently infected monkey Vero cells. By contrast, IFN had no clear protective effect on either de novo or persistent BDV infections of rat C6 glioblastoma cells. IFN protected C6 cells from the cytopathic effects of vesicular stomatitis virus, excluding the possibility that these cells are devoid of a functional IFN system. In primary rat fibroblasts and in a human oligodendroglial cell line, IFN induced an efficient antiviral state against BDV. These results indicate that BDV is highly susceptible to the antiviral effect of IFN in some cell lines, while others seem to lack undefined components of the IFN system which mediate protection against BDV.
300 million years of conserved synteny between chicken Z and human chromosome 9.
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Borna disease virus in human brains with a rare form of hippocampal degeneration but not in brains of patients with common neuropsychiatric disorders.
To estimate the frequency of persistent Borna disease virus (BDV) infections of the human central nervous system and to determine which neuropsychiatric disorders might be associated with this viral infection, reverse transcription-nested polymerase chain reaction was used to screen a large collection of autopsy brain samples for the presence of BDV-specific nucleic acids. The presence of BDV RNA was found in 3 brains of persons with psychiatric symptoms and prominent hippocampal degeneration previously reported to be positive by others. However, no BDV RNA was detected in 86 randomly collected brains from persons with various psychiatric disorders, including schizophrenia, affective disorders, and Alzheimer's disease, or from suicide victims or in 52 brains from healthy controls. Furthermore, no BDV-RNA was detected in 16 surgical brain samples from persons with epilepsy-associated hippocampal sclerosis. These results indicate that life-long persistent BDV infections are rare in humans and that such infections may be associated with certain forms of hippocampal degeneration.
Elimination of duck hepatitis B virus RNA-containing capsids in duck interferon-alpha-treated hepatocytes.
Evidence is presented that the previously cloned type I duck interferon (DuIFN) cDNA encodes a homologue of mammalian interferon-alpha (IFN-alpha). Recombinant DuIFN-alpha was used to study the inhibition of duck hepatitis B virus (DHBV) replication in primary hepatocytes in order to determine the IFN-sensitive steps of the virus replication cycle. IFN-treated cells accumulated two- to threefold-lower amounts of viral RNA transcripts early during infection, when IFN was added before virus. This reduction was not due to inhibition of virus entry since initial covalently closed circular DNA levels were not decreased in IFN-treated cells. Interestingly, the inhibitory effect of IFN on viral RNA levels was not observed in cells infected with a mutant DHBV that fails to synthesize core protein, suggesting that an uncharacterized core protein-mediated enhancing effect is blocked by IFN. When IFN was added at 4 days postinfection, encapsidated viral RNA pregenomes disappeared from infected cells within 3 days. This depletion was not simply due to conversion of pregenomes to DNA since depletion was not blocked by phosphonoformic acid, an inhibitor of the viral reverse transcriptase. The intracellular concentration of intact nucleocapsids was reduced, suggesting that in the presence of IFN pregenome-containing capsids were selectively depleted in hepatocytes. Thus, two steps in DHBV replication that involve the viral core protein were inhibited by DuIFN-alpha.
A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein.
Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta). Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography. Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.
Domains mediating intramolecular folding and oligomerization of MxA GTPase.
MxA is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses by a still poorly understood mechanism. Previous biochemical studies indicated that the C terminus of MxA folds back to form a functional GTP-binding pocket, and that an internal fragment contains a domain required for oligomerization. Using the yeast two-hybrid system, we have now mapped these domains. MxA sequences located downstream of amino acid 564 were found to strongly interact with an internal domain that includes amino acids 372 to 540. This interaction was abolished by mutating phenylalanine 382 or leucine 612, which is part of a leucine zipper motif. Neither the C-terminal nor the internal MxA fragments formed homo-oligomers. Using a mammalian nuclear transport assay that can detect protein-protein interactions, we further found that full-length MxA forms complexes with MxA fragments that include amino acids 372 to 540. This interaction was not observed when phenylalanine 382 was exchanged for alanine or arginine. Furthermore, interaction of two full-length MxA molecules occurred only if at least one of them carried a functional C-terminal leucine zipper motif. These results suggest that C-terminal back-folding and oligomerization are two alternative outcomes of the same type of interaction between the C-terminal and the internal domains of MxA. Intramolecular interaction is believed to result in the formation of MxA monomers, whereas intermolecular interaction may induce the formation of large MxA oligomers.
[Borna disease virus and psychiatric diseases. Fact or artefact?].
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Vaccinia virus-encoded cytokine receptor binds and neutralizes chicken interferon-gamma.
To counteract the host immune response, poxviruses have evolved secreted factors that bind cytokines and thereby neutralize their biological activities. The vaccinia virus B8R gene encodes a protein that neutralizes interferon-gamma (IFN-gamma) from several mammals including man, cow, rat, and rabbit but not mice. We now report that the activity of the B8R gene product is not restricted to cytokines of mammals: it also efficiently neutralized chicken IFN-gamma. B8R blocked chicken IFN-gamma-mediated induction of guanylate binding protein RNA in the chicken fibroblast cell line CEC-32 and secretion of nitric oxide in HD-11 cells. Radiolabeled baculovirus-expressed B8R efficiently bound to immobilized recombinant chicken IFN-gamma. Scatchard analysis revealed a binding constant of chicken IFN-gamma to B8R of approximately 0.5 nM. A mutant form of chicken IFN-gamma which lacks the 18 C-terminal amino acids and which has lost more than 99% of its biological activity was able to block the IFN-gamma-neutralizing effect of B8R. Binding studies showed that the mutant protein bound radiolabeled B8R only about threefold less well than wild-type chicken IFN-gamma but failed to compete with wild-type chicken IFN-gamma for binding to the cellular receptor. These results suggest that the extreme C terminus of chicken IFN-gamma is crucial for binding to its cellular receptor but less important for recognition by the viral cytokine receptor.
Promoter structures and differential responses to viral and nonviral inducers of chicken type I interferon genes.
Two serologically distinct type I interferons (IFNs), designated ChIFN1 and ChIFN2, are known in the chicken. ChIFN1 is encoded by a family of 10 or more genes, whereas ChIFN2 is encoded by a single gene. We show here that ChIFN1 and ChIFN2 transcripts are both strongly induced by Newcastle disease virus in primary chicken macrophages. By contrast, oral administration of the imidazoquinoline S-28463, which selectively induces IFN-alpha in mammals, led to a rapid accumulation of ChIFN1 (but not ChIFN2) transcripts in adult chicken spleen and thymus. The 5'-upstream region of the ChIFN2 gene contains a NF-kappaB consensus motif flanked by a sequence element that could serve as a binding site for transcription factor IRF-1, reminiscent of mammalian IFN-beta promoters, and it mediated powerful virus inducibility in a duck fibroblast cell line when cloned in front of a promoterless luciferase reporter gene. The 5'-upstream region of the cloned ChIFN1 gene contains two putative binding sites for IRF-1, but lacks NF-kappaB-binding sites, and it did not respond well to virus in transfected cells. Thus, the promoters of ChIFN1 and ChIFN2 genes not only exhibited differential responses to nonviral inducers in vivo, but also differed in structure and response to virus in transfected cells. These findings indicate that ChIFN2 represents the avian homolog of mammalian IFN-beta, whereas ChIFN1 seems to correspond to mammalian IFN-alpha.