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Georg Kochs - One of the best experts on this subject based on the ideXlab platform.
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Viral targeting of TFIIB impairs de novo polymerase II recruitment and affects antiviral immunity
2018Co-Authors: Darya A. Haas, Georg Kochs, Carola Vogt, Ellen Preuss, Arno Meiler, Katharina Geiger, Andreas PichlmairAbstract:Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxoVirus Thogoto Virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.
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Thogoto Virus ML protein inhibits IFN-α/β production by interacting with TFIIB.
2018Co-Authors: Darya A. Haas, Georg Kochs, Carola Vogt, Ellen Preuss, Arno Meiler, Katharina Geiger, Andreas PichlmairAbstract:A) Volcano plot of proteins enriched in ML vs. M pulldown in HEK293 cells and identified by AP-LC-MS/MS. HA-tagged M or ML proteins were overexpressed in 4 biological replicates. B) Schematic representation of ML protein and its mutants not binding TFIIB or CAPN15. C) IP of GST-tagged M or ML (wt and mut) and co-IP of FLAG-tagged CAPN15 and TFIIB transiently overexpressed in HEK293 cells. Western blot is a representative of two independent experiments with similar results. D) IFN-α/β levels after infection with THOV wt, ΔML or mutant ML(SW) 24 h.p.i. SN from infected HeLa cells were applied to 293T Mx1-luc. *—p value < 0.05, NS–non-significant. Bar graph shows mean with SD of three technical replicates and is a representative of four independent experiments with similar results. Significance was estimated with Kruskal-Wallis test with Dunn’s multiple comparison post-test.
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Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like MyxoVirus Resistance Protein A for GTPase Activation and Antiviral Activity
The Journal of biological chemistry, 2015Co-Authors: Alexej Dick, Georg Kochs, Song Gao, Alexander Von Der Malsburg, Laura Graf, Daniel Olal, Oliver DaumkeAbstract:MyxoVirus resistance (Mx) GTPases are induced by interferon and inhibit multiple Viruses, including influenza and human immunodeficiency Viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto Virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse Viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action.
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evolution guided identification of antiviral specificity determinants in the broadly acting interferon induced innate immunity factor mxa
Cell Host & Microbe, 2012Co-Authors: Patrick S Mitchell, Otto Haller, Corinna Patzina, Michael Emerman, Harmit S Malik, Georg KochsAbstract:MxA is an interferon-induced dynamin-like GTPase with wide-ranging antiviral activity, which hinges upon detection of unique viral structures that differ across Virus families. Despite elucidation of its structure, the basis of MxA antiviral specificity remains enigmatic. We used an evolution-guided approach to identify the loop L4 of MxA as a hotspot for recurrent positive selection in primates. Further, we show that single amino acid changes in L4 are necessary and sufficient to explain dramatic differences in species-specific antiviral activity of primate MxA proteins against the orthomyxoViruses Thogoto Virus and influenza A Virus. Taken together, our findings identify a genetic determinant of MxA target recognition and suggest a model by which MxA achieves antiviral breadth without compromising viral specificity.
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Thogoto Virus ml protein is a potent inhibitor of the interferon regulatory factor 7 transcription factor
Journal of General Virology, 2010Co-Authors: Nico Buettner, Carola Vogt, Friedemann Weber, Luis Martinezsobrido, Zoe Waibler, Georg KochsAbstract:The tick-transmitted orthomyxoVirus Thogoto Virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.
Otto Haller - One of the best experts on this subject based on the ideXlab platform.
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evolution guided identification of antiviral specificity determinants in the broadly acting interferon induced innate immunity factor mxa
Cell Host & Microbe, 2012Co-Authors: Patrick S Mitchell, Otto Haller, Corinna Patzina, Michael Emerman, Harmit S Malik, Georg KochsAbstract:MxA is an interferon-induced dynamin-like GTPase with wide-ranging antiviral activity, which hinges upon detection of unique viral structures that differ across Virus families. Despite elucidation of its structure, the basis of MxA antiviral specificity remains enigmatic. We used an evolution-guided approach to identify the loop L4 of MxA as a hotspot for recurrent positive selection in primates. Further, we show that single amino acid changes in L4 are necessary and sufficient to explain dramatic differences in species-specific antiviral activity of primate MxA proteins against the orthomyxoViruses Thogoto Virus and influenza A Virus. Taken together, our findings identify a genetic determinant of MxA target recognition and suggest a model by which MxA achieves antiviral breadth without compromising viral specificity.
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interferon induced mx proteins in antiviral host defense
Biochimie, 2007Co-Authors: Otto Haller, Peter Staeheli, Georg KochsAbstract:Abstract Mx proteins are key components of the antiviral state induced by interferons in many species. They belong to the class of dynamin-like large guanosine triphosphatases (GTPases) known to be involved in intracellular vesicle trafficking and organelle homeostasis. Mx GTPases share structural and functional properties with dynamin, such as self-assembly and association with intracellular membranes. A unique property of some Mx GTPases is their antiviral activity against a wide range of RNA Viruses, including influenza Viruses and members of the bunyaVirus family. These Viruses are inhibited at an early stage in their life cycle, soon after host cell entry and before genome amplification. The mouse Mx1 GTPase accumulates in the cell nucleus where it associates with components of the PML nuclear bodies and inhibits influenza and Thogoto Viruses known to replicate in the nucleus. The human MxA GTPase accumulates in the cytoplasm and is partly associated with a COP-I-positive subcompartment of the endoplasmic reticulum. This membrane compartment seems to provide an interaction platform that facilitates viral target recognition. In the case of bunyaViruses, MxA recognizes the viral nucleocapsid protein and interferes with its role in viral genome replication. In the case of Thogoto Virus, MxA recognizes the viral nucleoprotein and prevents the incoming viral nucleocapsids from being transported into the nucleus, the site of viral transcription and replication. In both cases, GTP-binding and carboxy-terminal effector functions of MxA are required for target recognition. In general, Mx GTPases appear to detect viral infection by sensing nucleocapsid-like structures. As a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new Virus particles.
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interferon induced mx proteins dynamin like gtpases with antiviral activity
Traffic, 2002Co-Authors: Otto Haller, Georg KochsAbstract:Mx proteins are interferon-induced GTPases that belong to the dynamin superfamily of large GTPases. Similarities include a high molecular weight, a propensity to self-assemble, a relatively low affinity for GTP, and a high intrinsic rate of GTP hydrolysis. A unique property of Mx GTPases is their antiviral activity against a wide range of RNA Viruses, including bunya- and orthomyxoViruses. The human MxA GTPase accumulates in the cytoplasm of interferon-treated cells, partly associating with the endoplasmic reticulum. In the case of bunyaViruses, MxA interferes with transport of the viral nucleocapsid protein (N) to the Golgi compartment, the site of Virus assembly. In the case of Thogoto Virus (an orthomyxoVirus), MxA prevents the incoming viral nucleocapsids from being transported into the nucleus, the site of viral transcription and replication. In both cases, the GTP-binding and carboxy-terminal effector functions of MxA are required for target recognition. In general, Mx GTPases appear to detect viral infection by sensing nucleocapsid-like structures. As a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new Virus particles.
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interferon induced antiviral mx1 gtpase is associated with components of the sumo 1 system and promyelocytic leukemia protein nuclear bodies
Experimental Cell Research, 2001Co-Authors: Othmar G Engelhardt, Georg Kochs, Evelyn Ullrich, Otto HallerAbstract: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.
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Formation of Virus-like particles from cloned cDNAs of Thogoto Virus
2000Co-Authors: Elke Wagner, Othmar G Engelhardt, Otto Haller, Friedemann Weber, Georg KochsAbstract:Thogoto Virus (THOV) is the type species of tick-transmitted orthomyxoViruses. Here, we describe the generation of Virus-like particles (VLP) of THOV from cloned cDNAs. To synthesize the six structural proteins of THOV in mammalian cells, we used T7-controlled expression plasmids and a recombinant vaccinia Virus producing T7 RNA polymerase. A minireplicon encoding a reporter gene flanked by THOV promoter sequences was expressed by the cellular RNA polymerase I. The recombinant pro-teinswere functional in encapsidation, amplification and transcription of the minireplicon RNA. Fur-thermore, the artificial nucleocapsids were packaged into THO-VLPs that transferred the minireplicon to indicator cells. This system should be helpful in generating recombinant THOV entirely from cloned cDNAs. Thogoto Virus (THOV) is the type species of a newly established genus of tick-transmitted Viruses in the family Orthomyxoviridae (Pringle, 1996). The genome of THOV consists of six single-stranded RNA segments of negative polarity (vRNA; Clerx et al., 1983). Each segment encodes one of the six structural proteins: the three subunits of the viral RNA polymerase complex (PB1, PB2 and PA; Leahy et al.
Jovan Pavlovic - One of the best experts on this subject based on the ideXlab platform.
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Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections
Journal of virology, 1999Co-Authors: Hans Peter Hefti, Otto Haller, Michael Frese, Heinrich Landis, Claudio Di Paolo, Adriano Aguzzi, Jovan PavlovicAbstract: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.
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human mxa protein protects mice lacking a functional alpha beta interferon system against la crosse Virus and other lethal viral infections
Journal of Virology, 1999Co-Authors: Hans Peter Hefti, Otto Haller, Michael Frese, Heinrich Landis, Claudio Di Paolo, Adriano Aguzzi, Jovan PavlovicAbstract: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.
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human mxa protein confers resistance to semliki forest Virus and inhibits the amplification of a semliki forest Virus based replicon in the absence of viral structural proteins
Journal of Virology, 1998Co-Authors: Heinrich Landis, Hans Peter Hefti, Claudio Di Paolo, Angela Simonjodicke, Andreas Kloti, J J Schnorr, Sibylle Schneiderschaulies, Jovan PavlovicAbstract:SFV is a member of the family Togaviridae (genus AlphaVirus), a family of mosquito-borne, positive-strand RNA Viruses which has a large host range and whose most common complication is encephalitis (17). The Virus enters the cells via receptor-mediated endocytosis (22). The uncoating of the nucleocapsids depends on ribosomes which release the capsid proteins from the nucleocapsid and sequester them (35). In contrast to negative-strand RNA Viruses, which transport their RNA transcriptase within the virion into the cells, the liberated genomic 49S RNA of Semliki Forest Virus (SFV) serves directly as mRNA for the synthesis of the RNA polymerase. For replication, which occurs in the cytoplasm, the parental 49S positive-strand RNA is transcribed into a 49S negative-strand RNA, which in turn serves as a template for either the synthesis of progeny 49S positive-strand genomic RNA or subgenomic 26S mRNA directing the synthesis of structural proteins (15). SFV infection is strongly impaired in mice following treatment with type I interferon (IFN) (7). IFN also mediates a very potent activity against SFV replication in cell cultures, and the Virus is widely used as challenging agent in Virus yield reduction assays (20). IFN-α treatment leads to reduced viral protein levels and hinders Virus-mediated host shutoff (24). However, little is known about the molecular mechanisms of this antiviral action. The antiviral effect of IFNs is mediated by several IFN-induced proteins which inhibit the multiplication of Viruses by distinct mechanisms (for reviews see references 31 and 37). Some members of the Mx protein family were shown to contribute to this antiviral state by inhibiting the multiplication of different negative-strand RNA Viruses (5, 6, 8, 16, 23, 27, 33, 39, 43, 44, 46). The Mx proteins form a small group of GTPases (9, 25, 29) and are synthesized under the stringent control of IFN type I (1, 38). The molecular mechanism of Mx action still remains unclear, but the GTPase activity appears to be essential for antiviral function (28). The antiviral properties of Mx proteins differ and are influenced by the intracellular localization of a particular Mx protein (14, 45, 47). Murine Mx1, which accumulates in the nucleus (4, 11), appears to be specific for Orthomyxoviridae (8, 27, 39, 43). The protein interferes with influenza Virus replication at the level of primary transcription (18, 19, 26), suggesting an interaction with the viral polymerase complex. Indeed, overexpression of PB2, a subunit of the influenza Virus polymerase complex, leads to a partial neutralization of the antiviral effect of Mx1 (12, 41). The human MxA protein, which accumulates in the cytoplasm, has a broader activity, inhibiting the multiplication of influenza Virus, Thogoto Virus (Orthomyxoviridae), vesicular stomatitus Virus (VSV) (Rhabdoviridae), measles Virus (MV), human parainfluenza Virus type 3 (Paramyxoviridae), and several members of the family Bunyaviridae (5, 6, 16, 27, 32, 33, 44). In contrast to Mx1, MxA appears to block the multiplication of influenza Virus at a poorly defined cytoplasmic step following primary transcription (26). In the case of VSV, MxA inhibits primary transcription (40). For MV, the situation is clearly different. First, the protective effect of MxA against MV was detected only in the human monocytic cell line U937 and in the glioblastoma cell line U87. Furthermore, MxA inhibited the multiplication of MV at the level of either viral RNA synthesis or synthesis of viral glycoproteins, depending on the cell line used (32, 33). We report here that the antiviral specificity of MxA is extended to SFV, a positive-strand RNA Virus. The activity of MxA against SFV appears to be either cell type or species specific, since no inhibitory effect was found in MxA-transfected mouse 3T3 fibroblasts (27). The fact that the accumulation of viral RNAs and proteins was inhibited points to a block occurring early in the replicative cycle. In order to define potential viral targets of MxA, we took advantage of an SFV replicon-based vector coding only for the viral replicase. The viral structural genes are replaced by the bacterial LacZ reporter gene (21). Upon transfection into cells, vector-derived recombinant RNA is amplified by virtue of its self-encoded replicase, and as a consequence large quantities of β-galactosidase (β-Gal) are produced. In MxA-transfected HEp-2 cells but not in mouse 3T3 cells, expression of β-Gal was dramatically reduced. These results demonstrate that the SFV structural proteins are not the target of MxA action and further suggest the involvement of species-specific cellular factors.
Friedemann Weber - One of the best experts on this subject based on the ideXlab platform.
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evolution and antiviral specificities of interferon induced mx proteins of bats against ebola influenza and other rna Viruses
Journal of Virology, 2017Co-Authors: Jonas Fuchs, Martin Holzer, Mirjam Schilling, Corinna Patzina, Andreas Schoen, Thomas Hoenen, Gert Zimmer, Manja Marz, Friedemann WeberAbstract:Bats serve as a reservoir for various, often zoonotic Viruses, including significant human pathogens such as Ebola- and influenza Viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxoVirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, Virus-like particles and Virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of Viruses circulating in bats, including Ebola- and influenza A-like Viruses. The related Thogoto Virus, however, which is not known to infect bats was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these Viruses in their bat hosts.
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Thogoto Virus ml protein is a potent inhibitor of the interferon regulatory factor 7 transcription factor
Journal of General Virology, 2010Co-Authors: Nico Buettner, Carola Vogt, Friedemann Weber, Luis Martinezsobrido, Zoe Waibler, Georg KochsAbstract:The tick-transmitted orthomyxoVirus Thogoto Virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.
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the interferon antagonist ml protein of Thogoto Virus targets general transcription factor iib
Journal of Virology, 2008Co-Authors: Carola Vogt, Ellen Preuss, Daniel C Mayer, Friedemann Weber, Martin Schwemmle, Georg KochsAbstract:The ML protein of Thogoto Virus, a tick-transmitted orthomyxoVirus, is a splice variant of the viral matrix protein and antagonizes the induction of antiviral type I interferon (IFN). Here we identified the general RNA polymerase II transcription factor IIB (TFIIB) as an ML-interacting protein. Overexpression of TFIIB neutralized the inhibitory effect of ML on IRF3-mediated promoter activation. Moreover, a recombinant Virus expressing a mutant ML protein unable to bind TFIIB was severely impaired in its ability to suppress IFN induction. We concluded that TFIIB binding is required for the IFN antagonist effect exerted by ML. We further demonstrate that the ML-TFIIB interaction has surprisingly little impact on gene expression in general, while a strong negative effect is observed for IRF3- and NF-κB-regulated promoters.
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Thogoto Virus ML protein suppresses IRF3 function.
Virology, 2005Co-Authors: Stephanie Jennings, Friedemann Weber, Luis Martínez-sobrido, Adolfo García-sastre, Georg KochsAbstract:The Thogoto Virus (THOV) is a member of the family Orthomyxoviridae. It prevents induction of alpha/beta interferons (IFN) in cell culture and in vivo via the action of the viral ML protein. Phenotypically, the effect of THOV ML resembles that of the NS1 protein of influenza A Virus (FLUAV) in that it blocks the expression of IFN genes. IFN expression depends on IFN regulatory factor 3 (IRF3). Upon activation, IRF3 forms homodimers and accumulates in the nucleus where it binds the transcriptional coactivator CREB-binding protein (CBP). Here, we show that expression of ML blocked the transcriptional activity of IRF3 after stimulation by Virus infection. Further biochemical analysis revealed that ML acts by blocking IRF3 dimerization and association with CBP. Surprisingly, however, ML did not interfere with the nuclear transport of IRF3. Thus, the action of ML differs strikingly from that of FLUAV NS1 that prevents IFN induction by retaining IRF3 in the cytoplasm.
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Formation of Virus-like particles from cloned cDNAs of Thogoto Virus
2000Co-Authors: Elke Wagner, Othmar G Engelhardt, Otto Haller, Friedemann Weber, Georg KochsAbstract:Thogoto Virus (THOV) is the type species of tick-transmitted orthomyxoViruses. Here, we describe the generation of Virus-like particles (VLP) of THOV from cloned cDNAs. To synthesize the six structural proteins of THOV in mammalian cells, we used T7-controlled expression plasmids and a recombinant vaccinia Virus producing T7 RNA polymerase. A minireplicon encoding a reporter gene flanked by THOV promoter sequences was expressed by the cellular RNA polymerase I. The recombinant pro-teinswere functional in encapsidation, amplification and transcription of the minireplicon RNA. Fur-thermore, the artificial nucleocapsids were packaged into THO-VLPs that transferred the minireplicon to indicator cells. This system should be helpful in generating recombinant THOV entirely from cloned cDNAs. Thogoto Virus (THOV) is the type species of a newly established genus of tick-transmitted Viruses in the family Orthomyxoviridae (Pringle, 1996). The genome of THOV consists of six single-stranded RNA segments of negative polarity (vRNA; Clerx et al., 1983). Each segment encodes one of the six structural proteins: the three subunits of the viral RNA polymerase complex (PB1, PB2 and PA; Leahy et al.
Hans Peter Hefti - One of the best experts on this subject based on the ideXlab platform.
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Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections
Journal of virology, 1999Co-Authors: Hans Peter Hefti, Otto Haller, Michael Frese, Heinrich Landis, Claudio Di Paolo, Adriano Aguzzi, Jovan PavlovicAbstract: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.
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human mxa protein protects mice lacking a functional alpha beta interferon system against la crosse Virus and other lethal viral infections
Journal of Virology, 1999Co-Authors: Hans Peter Hefti, Otto Haller, Michael Frese, Heinrich Landis, Claudio Di Paolo, Adriano Aguzzi, Jovan PavlovicAbstract: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.
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human mxa protein confers resistance to semliki forest Virus and inhibits the amplification of a semliki forest Virus based replicon in the absence of viral structural proteins
Journal of Virology, 1998Co-Authors: Heinrich Landis, Hans Peter Hefti, Claudio Di Paolo, Angela Simonjodicke, Andreas Kloti, J J Schnorr, Sibylle Schneiderschaulies, Jovan PavlovicAbstract:SFV is a member of the family Togaviridae (genus AlphaVirus), a family of mosquito-borne, positive-strand RNA Viruses which has a large host range and whose most common complication is encephalitis (17). The Virus enters the cells via receptor-mediated endocytosis (22). The uncoating of the nucleocapsids depends on ribosomes which release the capsid proteins from the nucleocapsid and sequester them (35). In contrast to negative-strand RNA Viruses, which transport their RNA transcriptase within the virion into the cells, the liberated genomic 49S RNA of Semliki Forest Virus (SFV) serves directly as mRNA for the synthesis of the RNA polymerase. For replication, which occurs in the cytoplasm, the parental 49S positive-strand RNA is transcribed into a 49S negative-strand RNA, which in turn serves as a template for either the synthesis of progeny 49S positive-strand genomic RNA or subgenomic 26S mRNA directing the synthesis of structural proteins (15). SFV infection is strongly impaired in mice following treatment with type I interferon (IFN) (7). IFN also mediates a very potent activity against SFV replication in cell cultures, and the Virus is widely used as challenging agent in Virus yield reduction assays (20). IFN-α treatment leads to reduced viral protein levels and hinders Virus-mediated host shutoff (24). However, little is known about the molecular mechanisms of this antiviral action. The antiviral effect of IFNs is mediated by several IFN-induced proteins which inhibit the multiplication of Viruses by distinct mechanisms (for reviews see references 31 and 37). Some members of the Mx protein family were shown to contribute to this antiviral state by inhibiting the multiplication of different negative-strand RNA Viruses (5, 6, 8, 16, 23, 27, 33, 39, 43, 44, 46). The Mx proteins form a small group of GTPases (9, 25, 29) and are synthesized under the stringent control of IFN type I (1, 38). The molecular mechanism of Mx action still remains unclear, but the GTPase activity appears to be essential for antiviral function (28). The antiviral properties of Mx proteins differ and are influenced by the intracellular localization of a particular Mx protein (14, 45, 47). Murine Mx1, which accumulates in the nucleus (4, 11), appears to be specific for Orthomyxoviridae (8, 27, 39, 43). The protein interferes with influenza Virus replication at the level of primary transcription (18, 19, 26), suggesting an interaction with the viral polymerase complex. Indeed, overexpression of PB2, a subunit of the influenza Virus polymerase complex, leads to a partial neutralization of the antiviral effect of Mx1 (12, 41). The human MxA protein, which accumulates in the cytoplasm, has a broader activity, inhibiting the multiplication of influenza Virus, Thogoto Virus (Orthomyxoviridae), vesicular stomatitus Virus (VSV) (Rhabdoviridae), measles Virus (MV), human parainfluenza Virus type 3 (Paramyxoviridae), and several members of the family Bunyaviridae (5, 6, 16, 27, 32, 33, 44). In contrast to Mx1, MxA appears to block the multiplication of influenza Virus at a poorly defined cytoplasmic step following primary transcription (26). In the case of VSV, MxA inhibits primary transcription (40). For MV, the situation is clearly different. First, the protective effect of MxA against MV was detected only in the human monocytic cell line U937 and in the glioblastoma cell line U87. Furthermore, MxA inhibited the multiplication of MV at the level of either viral RNA synthesis or synthesis of viral glycoproteins, depending on the cell line used (32, 33). We report here that the antiviral specificity of MxA is extended to SFV, a positive-strand RNA Virus. The activity of MxA against SFV appears to be either cell type or species specific, since no inhibitory effect was found in MxA-transfected mouse 3T3 fibroblasts (27). The fact that the accumulation of viral RNAs and proteins was inhibited points to a block occurring early in the replicative cycle. In order to define potential viral targets of MxA, we took advantage of an SFV replicon-based vector coding only for the viral replicase. The viral structural genes are replaced by the bacterial LacZ reporter gene (21). Upon transfection into cells, vector-derived recombinant RNA is amplified by virtue of its self-encoded replicase, and as a consequence large quantities of β-galactosidase (β-Gal) are produced. In MxA-transfected HEp-2 cells but not in mouse 3T3 cells, expression of β-Gal was dramatically reduced. These results demonstrate that the SFV structural proteins are not the target of MxA action and further suggest the involvement of species-specific cellular factors.