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Caracterização funcional da proteína MxA: estudo do seu envolvimento com a mauinaria de SUMOilação

Carvalho, Carlos Eduardo Brantis de
Fonte: Universidade Estadual Paulista (UNESP) Publicador: Universidade Estadual Paulista (UNESP)
Tipo: Tese de Doutorado Formato: 295 f. : il.
POR
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Pós-graduação em Biotecnologia - IQ; The human MxA protein is a member of the superfamily of GTPases dynamins. The so called Mx proteins are present in the majority of the vertebrate organisms investigated so far and contain two structural domains, named GTPase and CID-GED (stalk), besides the capabilities of homo-oligomerization and association with intracellular membranes. Moreover, the Mx proteins are strictly produced upon cell sensibilization with type I and III interferons. The vast antiviral activity against RNA and DNA viruses, including the Influenza virus and members of the bunyaviridae family, is among the functional properties of MxA. Moreover, MX1 epigenetic silencing is associated with cellular immortalization in neoplasias. Therefore, the study of MxA is of great interest as it is a key component of the Interferon-mediated pathways and cell proliferation control. Recentely, in a two-hybrid screen using a fetal brain cDNA library, it was possible to reveal that MxA interacts with proteins related to the post-translational modification process named SUMOylation and to the assemble of the nuclear bodies named PML-NBs and with proteins implicated in the control of transcription and apoptose. In this study, it was investigated the interaction between MxA and the components of the protein SUMOylation pathway. It was possible to confirm the physical interaction between MxA and Ubc9 and SUMO1...

MxA GTPase Blocks Reporter Gene Expression of Reconstituted Thogoto Virus Ribonucleoprotein Complexes

Weber, Friedemann; Haller, Otto; Kochs, Georg
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2000 EN
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28.02%
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.

A Monomeric GTPase-Negative MxA Mutant with Antiviral Activity

Janzen, Christian; Kochs, Georg; Haller, Otto
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2000 EN
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28.06%
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.

Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections

Hefti, Hans Peter; Frese, Michael; Landis, Heinrich; Di Paolo, Claudio; Aguzzi, Adriano; Haller, Otto; Pavlovic, Jovan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/1999 EN
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28.15%
The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-α/β). 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 β subunit of the IFN-α/β 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...

Inhibition of Hepatitis B Virus Replication by the Interferon-Inducible MxA Protein

Gordien, Emmanuel; Rosmorduc, Olivier; Peltekian, Cécile; Garreau, Florianne; Bréchot, Christian; Kremsdorf, Dina
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2001 EN
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28.13%
Human MxA is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several RNA viruses. We had previously shown that overexpression of the hepatitis B virus (HBV) capsid led to selective downregulation of MxA gene expression, suggesting a mechanism by which the virus escapes from the host defense system (O. Rosmorduc, H. Sirma, P. Soussan, E. Gordien, P. Lebon, M. Horisberger, C. Brechot and D. Kremsdorf, J. Gen. Virol. 80:1253–1262, 1999). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two- to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed...

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

Kochs, Georg; Janzen, Christian; Hohenberg, Heinz; Haller, Otto
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 05/03/2002 EN
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28.02%
Bunyaviruses replicate in the cytoplasm of infected cells. New viral particles are formed by budding of nucleocapsids into the Golgi apparatus. We have previously shown that the IFN-induced human MxA protein inhibits bunyavirus replication by an unknown mechanism. Here we demonstrate that MxA binds to the nucleocapsid protein of La Crosse virus (LACV) and colocalizes with the viral protein in cytoplasmic complexes. Electron microscopy revealed that these complexes accumulated in the perinuclear area and consisted of highly ordered fibrillary structures. A similar MxA-mediated redistribution of viral nucleocapsid proteins was detected with other bunyaviruses, such as Bunyamwera virus and Rift Valley fever virus. MxA(E645R), a carboxy-terminal mutant of MxA without antiviral activity against LACV, did not lead to complex formation. Wild-type MxA, but not MxA(E645R), was able to bind to LACV nucleocapsid protein in coimmunoprecipitation assays, demonstrating the importance of the carboxy-terminal effector domain of MxA. These results illustrate an efficient mechanism of IFN action whereby an essential virus component is trapped in cytoplasmic inclusions and becomes unavailable for the generation of new virus particles.

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

Landis, Heinrich; Simon-Jödicke, Angela; Klöti, Andreas; Di Paolo, Claudio; Schnorr, Jens-Jörg; Schneider-Schaulies, Sibylle; Hefti, Hans Peter; Pavlovic, Jovan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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28.13%
Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins...

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Ponten, A; Sick, C; Weeber, M; Haller, O; Kochs, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1997 EN
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Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus...

Cell type-specific MxA-mediated inhibition of measles virus transcription in human brain cells.

Schneider-Schaulies, S; Schneider-Schaulies, J; Schuster, A; Bayer, M; Pavlovic, J; ter Meulen, V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1994 EN
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27.98%
Measles virus (MV)-specific transcription in human brain cells is characterized by particularly low abundances of the distal mRNAs encoding the MV envelope proteins. Similar transcriptional restrictions of the closely related vesicular stomatitis virus have been observed in mouse fibroblasts constitutively expressing the interferon-inducible MxA protein (P. Staeheli and J. Pavlovic, J. Virol. 65:4498-4501, 1991). We found that MV infection of human brain cells is accompanied by rapid induction and high-level expression of endogenous MxA proteins. After stable transfection of MxA, human glioblastoma cells (U-87-MxA) released 50- to 100-fold less infectious virus and expression of viral proteins was highly restricted. The overall MV-specific transcription levels were reduced by up to 90%, accompanied by low relative frequencies of the distal MV-specific mRNAs. These restrictions were linked to an inhibition of viral RNA synthesis and not to a decreased stability of the viral RNAs. Our results indicate that expression of MxA is associated with transcriptional attenuation of MV in brain cells, thus probably contributing to the establishment of persistent MV central nervous system infections. In addition, the mechanism of MxA-dependent resistance against MV infection...

MxA-dependent inhibition of measles virus glycoprotein synthesis in a stably transfected human monocytic cell line.

Schnorr, J J; Schneider-Schaulies, S; Simon-Jödicke, A; Pavlovic, J; Horisberger, M A; ter Meulen, V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1993 EN
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28.06%
The alpha/beta (type I) interferon-inducible human MxA protein confers resistance to vesicular stomatitis virus (VSV) and influenza A virus in MxA-transfected mouse 3T3 cells (3T3/MxA). We investigated the inhibitory effects of the MxA protein on measles virus (MV) and VSV in the human monocytic cell line U937. In transfected U937 clones which constitutively express MxA (U937/MxA), the release of infectious MV and VSV was reduced approximately 100-fold in comparison with control titers. Transcription of VSV was inhibited similar to that observed for 3T3/MxA cells, whereas no difference was detected for MV in the rates of transcription or the levels of MV-specific mRNAs. In contrast, analysis of MV protein expression by immunofluorescence and immunoprecipitation revealed a significant reduction in the synthesis of MV glycoproteins F and H in U937/MxA cells. These data demonstrate a virus-specific effect of MxA which may, in the case of MV, contribute to the establishment of a persistent infection in human monocytic cells.

Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins.

Horisberger, M A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1992 EN
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MxA is an abundant and ubiquitous cytoplasmic protein induced by alpha/beta interferon in human cells. Upon full induction, it can constitute 0.5 to 1% of cytosolic proteins. MxA can bind elements of the cytoskeleton, such as actin and tubulins, and several larger cellular proteins. However, these protein-protein interactions seem to be transitory. The human MxA protein contains a tripartite GTP-binding domain consisting of GxxxxGKS, DxxG, and TKxD, where x is any amino acid. It is shown here that the native MxA protein has GTPase activity (GTP----GDP) when purified by immunoprecipitation with affinity-purified polyclonal antibodies directed against the C-terminal domain of MxA. The GTPase activity is greatly diminished by polyclonal antibodies directed against the N-terminal domain of MxA (the domain which contains the GTP-binding consensus elements). Amino acid substitution within the GTP-binding domain abolished the GTPase activity of the mutated MxA protein expressed in transfected CHO cells. The reaction is specific for GTP, and the approximate Km is 0.1 mM. The reaction has an absolute requirement for Mg2+. The turnover number is approximately 70 molecules of GTP hydrolyzed per min per MxA molecule. It is suggested that the human MxA protein has certain characteristics of the stress proteins.

Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

Turan, Kadir; Mibayashi, Masaki; Sugiyama, Kenji; Saito, Shoko; Numajiri, Akiko; Nagata, Kyosuke
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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28.15%
Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further...

Human MxA Protein Inhibits the Replication of Crimean-Congo Hemorrhagic Fever Virus

Andersson, Ida; Bladh, Linda; Mousavi-Jazi, Mehrdad; Magnusson, Karl-Eric; Lundkvist, Åke; Haller, Otto; Mirazimi, Ali
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2004 EN
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28.06%
Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.

Mechanism of human MxA protein action: variants with changed antiviral properties.

Zürcher, T; Pavlovic, J; Staeheli, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1992 EN
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28.06%
Cells respond to treatment with interferons by synthesizing several induced proteins, including one or more structurally related proteins collectively called Mx. Nuclear and cytoplasmic forms of Mx have been described, some of which inhibit virus replication. Human MxA is a cytoplasmic protein that specifically inhibits the multiplication of influenza virus and vesicular stomatitis virus. Here, we describe a mutant MxA protein, MxA(R645), which inhibited influenza virus but was inactive against vesicular stomatitis virus. It differs from wild-type MxA by a Glu to Arg substitution near the carboxy terminus. Like wild-type MxA, and as expected for an Mx protein acting in the cytoplasm, MxA(R645) blocked influenza virus at a step after primary transcription. When moved to the nucleus of transfected cells with the help of a foreign nuclear transport signal, its mode of action changed. Like mouse Mx1, nuclear MxA(R645) interfered with primary transcription of influenza virus, which is a nuclear process. Our results thus define an MxA region that determines antiviral specificity and further demonstrate that nuclear forms of MxA can mimic the action of mouse Mx1 whose natural location is the cell nucleus.

Induction of MxA Gene Expression by Influenza A Virus Requires Type I or Type III Interferon Signaling▿

Holzinger, Dirk; Jorns, Carl; Stertz, Silke; Boisson-Dupuis, Stéphanie; Thimme, Robert; Weidmann, Manfred; Casanova, Jean-Laurent; Haller, Otto; Kochs, Georg
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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28.09%
The human MxA gene belongs to the class of interferon (IFN)-stimulated genes (ISGs) involved in antiviral resistance against influenza viruses. Here, we studied the requirements for MxA induction by influenza A virus infection. MxA is transcriptionally upregulated by type I (alpha and beta) and type III (lambda) IFNs. Therefore, MxA is widely used in gene expression studies as a reliable marker for IFN bioactivity. It is not known, however, whether viruses can directly activate MxA expression in the absence of secreted IFN. By using an NS1-deficient influenza A virus and human cells with defects in IFN production or the STAT1 gene, we studied the induction profile of MxA by real-time reverse transcriptase PCR. The NS1-deficient virus is known to be a strong activator of the IFN system because NS1 acts as a viral IFN-antagonistic protein. Nevertheless, MxA gene expression was not inducible by this virus upon infection of IFN nonproducer cells and STAT1-null cells. Likewise, neither IFN-α nor IFN-λ had a sizeable effect on the STAT1-null cells, indicating that MxA expression requires STAT1 signaling and cannot be triggered directly by virus infection. In contrast, the expression of the IFN-stimulated gene ISG56 was induced by influenza virus in these cells...

Inhibition of Tumor Cell Motility by the Interferon-inducible GTPase MxA*S⃞

Mushinski, J. Frederic; Nguyen, PhuongMai; Stevens, Lisa M.; Khanna, Chand; Lee, Sunmin; Chung, Eun Joo; Lee, Min-Jung; Kim, Yeong Sang; Linehan, W. Marston; Horisberger, Michel A.; Trepel, Jane B.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 29/05/2009 EN
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28.15%
To identify pathways controlling prostate cancer metastasis we performed differential display analysis of the human prostate carcinoma cell line PC-3 and its highly metastatic derivative PC-3M. This revealed that a 78-kDa interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M cells. The gene encoding MxA, MX1, is located in the region of chromosome 21 deleted as a consequence of fusion of TMPRSS2 and ERG, which has been associated with aggressive, invasive prostate cancer. Stable exogenous MxA expression inhibited in vitro motility and invasiveness of PC-3M cells. In vivo exogenous MxA expression decreased the number of hepatic metastases following intrasplenic injection. Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX melanoma cells. A mutation in MxA that inactivated its GTPase reversed inhibition of motility and invasion in both tumor cell lines. Co-immunoprecipitation studies demonstrated that MxA associated with tubulin, but the GTPase-inactivating mutation blocked this association. Because MxA is a highly inducible gene, an MxA-targeted drug discovery screen was initiated by placing the MxA promoter upstream of a luciferase reporter. Examination of the NCI diversity set of small molecules revealed three hits that activated the promoter. In PC-3M cells...

Stalk Domain of the Dynamin-like MxA GTPase Protein Mediates Membrane Binding and Liposome Tubulation via the Unstructured L4 Loop*

von der Malsburg, Alexander; Abutbul-Ionita, Inbal; Haller, Otto; Kochs, Georg; Danino, Dganit
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
28.11%
The human MxA protein is an interferon-induced large GTPase with antiviral activity against a wide range of viruses, including influenza viruses. Recent structural data demonstrated that MxA oligomerizes into multimeric filamentous or ring-like structures by virtue of its stalk domain. Here, we show that negatively charged lipid membranes support MxA self-assembly. Like dynamin, MxA assembled around spherical liposomes inducing liposome tubulation. Cryo-transmission electron microscopy revealed that MxA oligomers around liposomes have a “T-bar” shape similar to dynamin. Moreover, biochemical assays indicated that the unstructured L4 loop of the MxA stalk serves as the lipid-binding moiety, and mutational analysis of L4 revealed that a stretch of four lysine residues is critical for binding. The orientation of the MxA molecule within the membrane-associated oligomer is in agreement with the proposed topology of MxA oligomers based on crystallographic data. Although oligomerization of wild-type MxA around liposomes led to the creation of helically decorated tubes similar to those formed by dynamin, this lipid interaction did not stimulate GTPase activity, in sharp contrast to the assembly-stimulated nucleotide hydrolysis observed with dynamin. Moreover...

Myxovirus Resistance Gene A (MxA) Expression Suppresses Influenza A Virus Replication in Alpha Interferon-Treated Primate Cells

Matzinger, Shannon R.; Carroll, Timothy D.; Dutra, Joseph C.; Ma, Zhong-Min; Miller, Christopher J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2013 EN
Relevância na Pesquisa
28.06%
Alpha interferon (IFN-α) production is triggered when influenza virus RNA is detected by appropriate pattern recognition receptors in the host cell. IFN-α induces the expression of more than 300 interferon-stimulated genes (ISGs), and this blunts influenza virus replication. The human ISG MxA can inhibit influenza A virus replication in mouse cells by interfering with a step in the virus replication cycle after primary transcription of the negative-strand RNA genome to mRNA (J. Pavlovic, O. Haller, and P. Staeheli, J. Virol. 66:2564–2569, 1992). To determine the role of MxA in blocking human influenza A virus replication in primate cells, we manipulated MxA expression in rhesus kidney epithelial cells (LLC-MK2) and human lung carcinoma cells (A549). We found that IFN-α treatment prior to influenza virus infection suppressed virus replication and induced the expression of many ISGs, including MxA. However, IFN-α-mediated suppression of virus replication was abolished by small interfering RNA (siRNA) knockdown of MxA expression in IFN-treated cells. In addition, influenza virus replication was suppressed in Vero cells stably transfected with MxA. A strand-specific reverse transcription-PCR (RT-PCR) assay showed that positive-strand influenza virus mRNA and negative-strand genomic RNA (gRNA) accumulated to high levels at 8 h after infection in control Vero cells containing the empty vector. However...

MxA interacts with and is modified by the SUMOylation machinery

Brantis-de-Carvalho, Carlos Eduardo; Maarifi, Ghizlane; Gonçalves Boldrin, Paulo Eduardo; Zanelli, Cleslei Fernando; Nisole, Sebastien; Chelbi-Alix, Mounira K.; Valentini, Sandro Roberto
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 151-163
ENG
Relevância na Pesquisa
38.13%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Processo FAPESP: 2003/09497-3; Processo FAPESP: 2010/50044-6; Mx proteins are evolutionarily conserved dynamin-like large GTPases involved in viral resistance triggered by types I and III interferons. The human MxA is a cytoplasmic protein that confers resistance to a large number of viruses. The MxA protein is also known to self-assembly into high molecular weight homo-oligomers. Using a yeast two-hybrid screen, we identified 27 MxA binding partners, some of which are related to the SUMOylation machinery. The interaction of MxA with Small-Ubiquitin MOdifier 1 (SUMO1) and Ubiquitin conjugating enzyme 9 (Ubc9) was confirmed by co-immunoprecipitation and co-localization by confocal microscopy. We identified one SUMO conjugation site at lysine 48 and two putative SUMO interacting motifs (SIMa and SIMb). We showed that MxA interacts with the EIL loop of SUMO1 in a SIM-independent manner via its CID-GED domain. The yeast two-hybrid mapping also revealed that Ubc9 binds to the MxA GTPase domain. Mutation in the putative SIMa and SIMb, which are located in the GTPase binding domain, reduced MxA antiviral activity. In addition...

Common variable immunodeficiency (CVID) and MxA-protein expression in blood leucocytes.

Rump, J A; Jakschiess, D; Walker, U; Schlesier, M; von Wussow, P; Peter, H H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1995 EN
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28.02%
The underlying immunopathogenic mechanism of CVID has been suspected to involve a chronic viral infection or an autoimmune condition. However, formal proof of viral infection is lacking. Measurement of MxA-protein in leucocyte lysates is a sensitive test for evaluating the activation of the host's interferon system. Both viral infections and autoimmune diseases such as systemic lupus erythematosus (SLE) strongly induce MxA-protein in peripheral leucocytes. We therefore examined 15 patients with longlasting hypogammaglobulinaemia for MxA-protein induction in vivo: 13 patients suffered from CVID, one from hyper-IgM syndrome, and one patient had chronic B lymphocytic leukaemia associated with immunoglobulin deficiency and chronic papilloma virus infection (condylomata accuminata). Only the latter patient exhibited a strong MxA-protein expression; two CVID patients were borderline positive, and the remaining 12 patients including the hyper-IgM syndrome were MxA-protein-negative. There was no relationship between MxA expression and low CD4/CD8 ratios or increased CD8/CD57+ T cell counts, although both conditions are often observed in CVID as well as in chronic viral infections. When exposed in vitro to interferon-alpha (IFN-alpha), peripheral blood leucocytes of four MxA-negative patients were capable of producing normal amounts of MxA-protein. Taken together...