Biomedical subjects

O Haller

Publications and source records attributed to O Haller.

At least 19 recordsLinked to original sources

PubMed2005

Chromosome evolution of MMU16 and RNO11: conserved synteny associated with gene order rearrangements explicable by intrachromosomal recombinations and neocentromere emergence.

Comparative mapping between the rat and mouse genomes has shown that some chromosomes are entirely or almost entirely conserved with respect to gene content. Such is the case of rat chromosome 11 (RNO11) and mouse chromosome 16 (MMU16). We determined to what extent such an extensive conservation of synteny is associated with a conserved gene order. Therefore, we regionally localized several genes on RNO11. The comparison of the gene map of RNO11 and MMU16 unambiguously shows that the gene order has not been conserved in the Murinae lineage, thereby implying the occurrence of intrachromosomal evolutionary rearrangements. The transition from one chromosome configuration to the other one can be explained either by two intrachromosomal recombinations or by a single intrachromosomal recombination accompanied by neocentromere emergence.

Animals↗

PubMed2001

Interferon-induced antiviral Mx1 GTPase is associated with components of the SUMO-1 system and promyelocytic leukemia protein nuclear bodies.

Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein.

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells.

Animals↗

PubMed2001

Interferon-induced rat Mx proteins confer resistance to Rift Valley fever virus and other arthropod-borne viruses.

Mx proteins belong to the interferon (IFN)-induced antiviral defense. The rat genome contains three Mx genes, ratMx1, ratMx2, and ratMx3. The Mx gene products differ in their subcellular localization and antiviral specificity. The nuclear ratMx1 protein confers resistance to influenza A virus, and the cytoplasmic ratMx2 is active against vesicular stomatitis virus (VSV), whereas the cytoplasmic ratMx3 protein is antivirally inactive. To investigate the antiviral potential of the rat Mx proteins against arboviruses, a phylogenetically diverse group of viruses that frequently infect rodents, we studied the replication of LaCrosse virus (LACV). Rift Valley fever virus (RVFV) (both family Bunyaviridae), and Thogoto virus (THOV) (family Orthomyxoviridae). To that end, we used transfected Vero cells constitutively expressing one of the rat Mx proteins. We observed that the antiviral activity of rat Mx proteins against these arboviruses correlates with their intracellular localization: ratMx1 is active against THOV, which replicates in the nucleus, whereas ratMx2 inhibits bunyaviruses that replicate in the cytoplasm. The results indicate that rats have evolved two Mx proteins to efficiently control viruses with different replication strategies.

Animals↗

PubMed2001

Rescue of recombinant Thogoto virus from cloned cDNA.

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.

Animals↗

PubMed2001

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

Animals↗

PubMed2000

Characterization of a novel serine/threonine kinase associated with nuclear bodies.

A novel protein kinase, Mx-interacting protein kinase (PKM), has been identified in a yeast two-hybrid screen for interaction partners of human MxA, an interferon-induced GTPase with antiviral activity against several RNA viruses. A highly conserved protein kinase domain is present in the N-terminal moiety of PKM, whereas an Mx interaction domain overlaps with C-terminal PEST sequences. PKM has a molecular weight of about 127,000 and exhibits high sequence homology to members of a recently described family of homeodomain-interacting protein kinases. Recombinant PKM has serine/threonine kinase activity that is abolished by a single amino acid substitution in the ATP binding domain (K221W). PKM catalyzes autophosphorylation and phosphorylation of various cellular and viral proteins. PKM is expressed constitutively and colocalizes with the interferon-inducible Sp100 protein and murine Mx1 in discrete nuclear structures known as nuclear bodies.

Amino Acid Sequence↗

PubMed2000

Inhibition of influenza C viruses by human MxA protein.

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.

Animals↗

PubMed2000

Constitutive expression of interferon-induced human MxA protein in transgenic tobacco plants does not confer resistance to a variety of RNA viruses.

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses, including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35 S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplication of tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.

GTP-Binding Proteins↗

PubMed2000

MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes.

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.

Animals↗

PubMed2000

A monomeric GTPase-negative MxA mutant with antiviral activity.

MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.

Amino Acid Substitution↗

PubMed2000

Thogoto virus matrix protein is encoded by a spliced mRNA.

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a segmented, negative-stranded RNA genome. In this study, we investigated the coding strategy of RNA segment 6 and found that it contains 956 nucleotides and codes for the matrix (M) protein. The full-length cDNA contains a single, long reading frame that lacks a stop codon but has coding capacity for a putative 35-kDa protein. In contrast, the M protein of THOV has an apparent molecular mass of 29 kDa as assessed by polyacrylamide gel electrophoresis. Therefore, we investigated the possibility of posttranscriptional processing of segment 6 transcripts by reverse transcription-PCR and identified a spliced mRNA that contains a stop codon and is translated into the 29-kDa M protein. Interestingly, the nontemplated UGA stop codon is generated by the splicing event itself. Thus, the unusual M coding strategy of THOV resembles that of Influenza C virus.

Amino Acid Sequence↗

PubMed1999

The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures.

To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule. mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N-terminal G domain. mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody-mediated crosslinking enhances GTP hydrolysis. In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation. Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo. mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions. These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition.

3T3 Cells↗

PubMed1999

Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.

3T3 Cells↗

PubMed1999

GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae).

Human MxA protein is an interferon-induced member of the dynamin superfamily of large GTPases. MxA inhibits the multiplication of several RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Previous studies have indicated that GTP binding is required for antiviral activity, but the mechanism of action is still unknown. Here, we have used an in vitro cosedimentation assay to demonstrate, for the first time, a GTP-dependent interaction between MxA GTPase and a viral target structure. The assay is based on highly active MxA GTPase as effector molecules, Thogoto virus nucleocapsids as viral targets, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) as a stabilizing factor. We show that MxA tightly interacts with viral nucleocapsids by binding to the nucleoprotein component. This interaction requires the presence of GTPgammaS and is mediated by domains in the carboxyl-terminal moiety of MxA. We propose that GTP-bound MxA adopts an antivirally active conformation that allows interaction with viral nucleocapsids, thereby impairing their normal function.

3T3 Cells↗

PubMed1999

PB2 polymerase subunit of Thogoto virus (Orthomyxoviridae family).

Thogoto virus (THOV) represents a new genus in the family Orthomyxoviridae. The three polymerase subunits PB1, PB2, and PA initiate transcription by a unique cap-stealing mechanism that involves the cleavage of only the m(7)GpppAm structure from cellular hnRNAs. Here, we report the cloning of the longest genomic segment of THOV coding for PB2. It comprises 2,375 nucleotides encoding a single large open reading frame flanked by the conserved terminal regions typical for orthomyxoviruses. The deduced amino acid sequence corresponds to a protein of 769 amino acids, with an estimated Mr of 88,042. Expression of the PB2 cDNA in mammalian cells revealed nuclear accumulation of the recombinant protein, consistent with the replication of THOV in the cell nucleus.

Amino Acid Sequence↗

PubMed1999

Identification of the murine Mx2 gene: interferon-induced expression of the Mx2 protein from the feral mouse gene confers resistance to vesicular stomatitis virus.

The mouse genome contains two related interferon-regulated genes, Mx1 and Mx2. Whereas Mx1 codes for the nuclear 72-kDa protein that interferes with influenza virus replication after interferon treatment, the Mx2 gene is nonfunctional in all laboratory mouse strains examined, since its open reading frame (ORF) is interrupted by an insertional mutation and a subsequent frameshift mutation. In the present study, we demonstrate that Mx2 mRNA of cells from feral mouse strains NJL (Mus musculus musculus) and SPR (Mus spretus) differs from that of the laboratory mouse strains tested. The Mx2 mRNA of the feral strains contains a single long ORF consisting of 656 amino acids. We further show that Mx2 protein in the feral strains is expressed upon interferon treatment and localizes to the cytoplasm much like the rat Mx2 protein, which inhibits vesicular stomatitis virus replication. Furthermore, transfected 3T3 cell lines of laboratory mouse origin expressing Mx2 from feral strains acquire slight resistance to vesicular stomatitis virus.

Amino Acid Sequence↗

PubMed1999

Human MxA protein protects mice lacking a functional alpha/beta interferon system against La crosse virus and other lethal viral infections.

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.

Alphavirus Infections↗