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T Michiels

Publications and source records attributed to T Michiels.

At least 19 recordsLinked to original sources
PubMed2001

The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production.

Theiler's virus is a picornavirus responsible for a persistent infection of the central nervous system of the mouse, leading to a chronic demyelinating disease considered to be a model for multiple sclerosis. The leader (L) protein encoded by Theiler's virus is a 76-amino-acid-long peptide containing a zinc-binding motif. This motif is conserved in the L proteins of all cardioviruses, including encephalomyocarditis virus. The L protein of Theiler's virus was suggested to interfere with the alpha/beta interferon (IFN-alpha/beta) response (W.-P. Kong, G. D. Ghadge, and R. P. Roos, Proc. Natl. Acad. Sci. USA 91:1796-1800, 1994). We show that expression of the L protein indeed inhibits the production of alpha/beta interferon by infected L929 cells. The L protein specifically inhibits the transcription of the IFN-alpha4 and IFN-beta genes, which are known to be activated early in response to viral infection. Mutation of the zinc finger was sufficient to block the anti-interferon activity, outlining the importance of this motif in the L protein function. In agreement with the anti-interferon role of the L protein, a virus bearing a mutation in the zinc-binding motif was dramatically impaired in its ability to persist in the central nervous system of SJL/J mice.

3T3 Cells↗
PubMed2000

Influence of the Theiler's virus L* protein on macrophage infection, viral persistence, and neurovirulence.

The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L*. We confirmed previous work showing that the L* ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L* protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L* ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L* AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L* initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605-8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.

Animals↗
PubMed1999

A coding RNA sequence acts as a replication signal in cardioviruses.

Theiler's virus and Mengo virus are representatives of the Cardiovirus genus within the picornavirus family. Their genome is an 8-kilobase long positive strand RNA molecule. This RNA molecule plays three roles in infected cells: It serves as a messenger RNA, acts as a template for genome replication, and is encapsidated to form progeny virions. We observed that a cis-acting signal required for replication of Theiler's virus was contained within a 130-nt stretch of the region encoding the capsid protein VP2. This RNA sequence does not influence internal ribosome entry site-mediated translation initiation and thus likely acts directly as a signal for the replication complex. We found a similar signal in the VP2-coding sequence of Mengo virus, and both signals could be functionally exchanged. Within the replication element, a 9-nt sequence that is highly conserved among cardioviruses was shown to be essential for replication. This conserved sequence was contained in mostly unpaired regions of the RNA secondary structure predicted for the replication elements of the various cardioviruses. Interestingly, a similar replication element has been reported to occur in the distantly related human rhinovirus type 14, suggesting that such elements could be conserved throughout the picornavirus family. However, the different location of the replication elements in rhinovirus and cardioviruses, and the fact that they were not functionally exchangeable, is raising intriguing questions about the evolution of such signals in picornaviruses.

Capsid↗
PubMed1999

Absence of internal ribosome entry site-mediated tissue specificity in the translation of a bicistronic transgene.

The 5' noncoding regions of the genomes of picornaviruses form a complex structure that directs cap-independent initiation of translation. This structure has been termed the internal ribosome entry site (IRES). The efficiency of translation initiation was shown, in vitro, to be influenced by the binding of cellular factors to the IRES. Hence, we hypothesized that the IRES might control picornavirus tropism. In order to test this possibility, we made a bicistronic construct in which translation of the luciferase gene is controlled by the IRES of Theiler's murine encephalomyelitis virus. In vitro, we observed that the IRES functions in various cell types and in macrophages, irrespective of their activation state. In vivo, we observed that the IRES is functional in different tissues of transgenic mice. Thus, it seems that the IRES is not an essential determinant of Theiler's virus tropism. On the other hand, the age of the mouse could be critical for IRES function. Indeed, the IRES was found to be more efficient in young mice. Picornavirus IRESs are becoming popular tools in transgenesis technology, since they allow the expression of two genes from the same transcription unit. Our results show that the Theiler's virus IRES is functional in cells of different origins and that it is thus a broad-spectrum tool. The possible age dependency of the IRES function, however, could be a drawback for gene expression in adult mice.

Animals↗
PubMed1999

Analysis of cellular mutants resistant to Theiler's virus infection: differential infection of L929 cells by persistent and neurovirulent strains.

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse and belongs to the Picornaviridae family. TMEV strains are divided into two subgroups on the basis of their pathogenicity. The first group contains two neurovirulent strains, FA and GDVII, which cause a rapid fatal encephalitis. The second group includes persistent strains, like DA and BeAn, which produce a biphasic neurological disease in susceptible mice. Persistence of these viruses in the white matter of the spinal cord leads to chronic inflammatory demyelination. L929 cells, which are susceptible to TMEV infection, were subjected to physicochemical mutagenesis. Cellular clones that became resistant to TMEV infection were selected by viral infection. Three such mutants resistant to strain GDVII were characterized to determine the step of the virus cycle that was inhibited. The mutation present in one of these mutant cell lines inhibited, by more than 1,000-fold, the entry of strain GDVII but hardly decreased infection by strain DA. In the two other cellular mutants, replication of the viral genome was slowed down. Interestingly, one of these mutant cell lines resisted infection by both the persistent and neurovirulent strains while the second cell line resisted infection by strain GDVII but remained susceptible to the persistent virus. These results show that although they have 95% identity at the amino acid sequence level, neurovirulent and persistent viruses use partly distinct pathways for both entry into cells and genome replication.

Animals↗
PubMed1998

Adaptation of Theiler's virus to L929 cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence.

Persistent strains of Theiler's virus, a murine picornavirus, produce a life-long infection of the central nervous system of the mouse and induce a chronic demyelinating disease. Strain DA1, a molecular clone of such a persistent strain, produces a prominent cytopathic effect in BHK-21 cells but is less efficient at infecting L929 cells. We cloned the cDNA of a derivative of virus DA1, adapted to promote a rapid cytopathic effect in L929 cells. Adaptation of the new variant (named KJ6) to L929 cells correlated with an enhanced viral entry rather than with an increased replication rate of the genome. Mutations responsible for L929 cells adaptation occurred in amino acids exposed at the surface of the capsid, in the CD loop of VP1 (100-102) and in the EF loop of VP2 (162-171-173), suggesting that these residues could be involved in receptor recognition. These two clusters of amino acids are precisely known to be part of neutralization epitopes. They also differentiate persistent from neurovirulent strains of Theiler's virus. Adaptation of the virus to L929 cells was accompanied by attenuation of its virulence for the mouse. Taken together, these data suggest a close relationship between receptor binding, virus neutralization, and virus phenotype.

Adaptation, Physiological↗
PubMed1997

Infection of macrophages by Theiler's murine encephalomyelitis virus is highly dependent on their activation or differentiation state.

Macrophages are the main targets of Theiler's murine encephalomyelitis virus (TMEV) during persistent demyelinating infection of mice. Replication of TMEV in macrophages was previously shown to depend on their activation state. Here, we show that the quality of the serum used for culture drastically influences viral entry in RAW264.7 macrophages.

Animals↗
PubMed1997

Protein 2A is not required for Theiler's virus replication.

Nonpolar mutations were introduced into all 12 regions of the genome of Theiler's murine encephalomyelitis virus. In agreement with data previously reported for other picornaviruses, mutations in regions 2B, 2C, 3A, 3B, 3C, and 3D totally abrogated viral RNA replication. Viruses with deletions in each of the capsid proteins retained RNA replication proficiency, although they were unable to propagate from cell to cell. As reported previously, mutations in the leader protein did not impair RNA replication or virus production in BHK-21 cells. Surprisingly, region 2A also appeared to be dispensable for the replication process. Indeed, up to 77 of the 133 amino acids of 2A could be deleted without significantly affecting RNA replication. 2A mutant viruses had only a slow cytopathic effect for BHK-21 cells and were totally avirulent for mice. As was the case for mutants lacking the leader protein, viruses with deletions in 2A propagated in BHK-21 cells, but their propagation was highly restricted in L929 cells.

Amino Acid Sequence↗
PubMed1995

Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus.

Most strains of Theiler's virus (TMEV) cause a persistent infection of the central nervous system of the mouse and a chronic demyelinating disease considered a model for multiple sclerosis. Two strains, on the contrary, cause an acute encephalitis and kill mice in a matter of days. We sequenced the leader and capsid coding region of three persistent (TO4, WW, and Yale) isolates and one neurovirulent (FA) isolate of TMEV. We compared these sequences and those already published for other isolates (DA, BeAn, GDVII, and Vilyuisk). The results suggest that virulent and persistent strains did not evolve as two separate groups, but rather that neurovirulent strains arose from a subgroup of persistent strains. The sequences of viruses isolated in different geographic areas and at different times were highly homologous, a surprising finding for an RNA virus. This suggests that severe constraints are imposed on the genome during the viral life cycle. The sequences of the TO4 and WW strains were identical, suggesting that the latter came from a laboratory contamination. The genomes of all the persistent strains sequenced so far contain an alternate open reading frame in the L region, which has been shown, in the case of the DA strain, to code for an 18-kDa protein called "I".

Amino Acid Sequence↗
PubMed1994

Individual chaperones required for Yop secretion by Yersinia.

Pathogenic yersiniae secrete anti-host proteins called Yops, by a recently discovered Sec-independent pathway. The Yops do not have a classical signal peptide at their N terminus and they are not processed during membrane translocation. The secretion domain is nevertheless contained in their N-terminal part but these domains do not resemble each other in the different Yops. We have previously shown that YopE secretion requires SycE, a 15-kDa acidic protein acting as a specific cytosolic chaperone. Here we show that the gene downstream from yopH encodes a 16-kDa acidic protein that binds to hybrid proteins made of the N-terminal part of YopH and either the bacterial alkaline phosphatase or the cholera toxin B subunit. Loss of this protein by mutagenesis led to accumulation of YopH in the cytoplasm and to a severe and selective reduction of YopH secretion. This protein thus behaves like the counterpart of SycE and we called it SycH. We also engineered a mutation in lcrH, the gene upstream from yopB and yopD, known to encode a 19-kDa acidic protein. Although this mutation was nonpolar, the mutant no longer secreted YopB and YopD. The product of lcrH could be immunoprecipitated together with cytoplasmic YopD. lcrH therefore seems to encode a YopD-specific chaperone, which we called SycD. Determination of the dependence of YopB on SycD requires further investigation. SycE, SycH, and SycD appear to be members of a new family of cytosolic chaperones required for Yop secretion.

Amino Acid Sequence↗
PubMed1994

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS.

Animals↗
PubMed1994

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.

Acute Disease↗
PubMed1991

ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions.

The virulence functions of Yersinia enterocolitica include the pYV-encoded Yop proteins and YadA adhesin as well as the chromosome-encoded enterotoxin, Yst. The yop and yadA genes form a temperature-activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes. The enterotoxin yst gene is silent in some collection strains including strain W22703. This paper describes two Tn5-Tc1 chromosomal insertion mutants of W22703 transcribing virF, and hence the yop and yadA genes, at low temperature. These mutants also resumed their production of Yst, with its typical temperature dependence. Both mutations were insertions in the same gene called ymoA for 'Yersinia modulator'. The cloned ymoA gene fully complemented the two mutations. Several properties of the mutants suggest that ymoA encodes a histone-like protein. According to the nucleic acid sequence, the product of ymoA is an 8064 Da protein rich in aspartic acid (9%), glutamic acid (9%) and lysine (10.5%), but the predicted amino acid sequence shows no similarity with any described histone-like protein. This work supports recent reports which propose a role for DNA topology and bacterial chromatin structure in thermoregulation of virulence functions.

Amino Acid Sequence↗
PubMed1991

Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica.

Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic yersiniae release large amounts of pYV plasmid-encoded proteins called Yops that are involved in pathogenesis. Yersinia enterocolitica also expresses an outer membrane protein that is considered an adhesin and called YadA (previously called P1 or YopA). The production of Yops is coordinately regulated by a 20-kb region of the plasmid referred to as the Ca2+ dependence region and containing at least four loci called virA, virB, virC, and virF. The virF gene encodes a key transcriptional activator of yop genes. We have shown here that virF is also required for transcription of yadA and that virB is necessary for full transcription of the yop and yadA genes. In contrast, mutations in genes virA and virC had only a weak influence on the transcription of yop and yadA genes. These mutations did not affect the production of YadA but they completely inhibited the translocation of Yops from the intracellular compartment to the extracellular milieu. We inferred from these data that virA and virC are involved in the specific transport of Yops. We analyzed the 8.5-kb virC region and showed that it is most probably a single operon containing 13 open reading frames called yscA to yscM (for Yop secretion). Protein YscC has a putative signal sequence and shares significant homology with outer membrane proteins involved in the secretion of pullulanase by Klebsiella pneumoniae (PulD) or in the assembly of filamentous bacteriophages (gene IV product). At least the putative products of yscD, yscJ, and yscL were shown to be required for the export of Yops. YscJ turned out to be YlpB, a lipoprotein that we had detected previously. The yscM gene shares homology with yopH, the adjacent gene on the pYV plasmid. Its product does not appear to be necessary for the production of Yops. Transcription of the virC operon was subjected to the same regulation as the yop genes.

Amino Acid Sequence↗
PubMed1991

Secretion of hybrid proteins by the Yersinia Yop export system.

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.

Amino Acid Sequence↗
PubMed1990

The pYV plasmid of Yersinia encodes a lipoprotein, YlpA, related to TraT.

A series of lipoproteins was detected in the membrane fraction of Yersinia enterocolitica W227, a typical strain from serotype O:9. At least two of them, YlpA and YlpB, are encoded by the pYV plasmid. The sequence of ylpA reveals the presence of a typical lipoprotein signal peptide. The mature YlpA protein would be 223 residues long with a calculated molecular weight of 23798 for the proteic moiety of the molecule. YlpA shares 88% identical residues with the TraT protein encoded by plasmid pED208, 80% identity with TraT proteins encoded by plasmids R100 and F, and 77% identity with the TraT protein encoded by the virulence plasmid of Salmonella typhimurium. The ylpA gene hybridized with the pYV plasmid of Yersinia pseudotuberculosis, suggesting that this gene is conserved among Yersinia spp. The production of YlpA is controlled by virF and only occurs at 37 degrees C in the absence of Ca2+ ions. This co-regulation with the yop genes suggests that ylpA is a virulence determinant. However, mutations in ylpA clearly affect neither the resistance to human serum nor the virulence for intravenously inoculated mice.

Amino Acid Sequence↗
PubMed1990

Secretion of Yop proteins by Yersiniae.

Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of the genus Yersinia cease growing and produce large amounts of a series of plasmid-encoded proteins involved in pathogenicity. These proteins, called Yops (for Yersinia outer membrane proteins), are detected in both the outer membrane fraction and the culture supernatant. We present here the nucleotide sequence of genes yop20 and yop25 from Yersinia enterocolitica O:9. Protein Yop25 is very similar to YopE, the corresponding protein from Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica O:8 (A. Forsberg and H. Wolf-Watz, J. Bacteriol. 172:1547-1555, 1990). This is the first report of a yop20 sequence of yersiniae. We present evidences that Yops are not membrane proteins. Their detection in the membrane fraction results either from copurification of large aggregates of extracellular Yops with the membrane fraction or from the adsorption of released proteins to the cell surface. In contrast with Yops, protein P1 has characteristics of a true membrane protein. The release of Yops by Y. enterocolitica occurs by a novel secretion mechanism that does not involve the cleavage of a typical signal sequence or the recognition of a carboxy-terminal domain.

Amino Acid Sequence↗