1 Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany
2 Heinrich-Pette-Institut für experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany
3 Molecular Biology Program, Department of Pathology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA
Correspondence
Othmar G. Engelhardt
othmar.engelhardt{at}path.ox.ac.uk
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ABSTRACT |
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Images showing the localization of mutant Mx1(C71S) are available as supplementary material in JGV Online.
Present address: Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK.
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INTRODUCTION |
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PML NBs, also known as ND10, Kr bodies or PML oncogenic domains, are dynamic subnuclear structures. Their composition, number and size depend on various stimuli and vary during the cell cycle. In recent years, an increasing number of proteins have been found to reside within or to be partially associated with these structures, which are organized by PML as the main structural component (Negorev & Maul, 2001). PML NBs seem to have numerous functions (Borden, 2002
; Piazza et al., 2001
; Salomoni & Pandolfi, 2002
; Zhong et al., 2000b
). They appear to be involved in the regulation of transcription, in apoptosis and in cell-cycle regulation. Furthermore, they have been proposed to be storage depots where proteins not needed at a given time are kept inactive or modified until they are recruited to their sites of action (Negorev & Maul, 2001
). Of particular interest is the role of PML NBs in viral infections. PML and other PML NB constituents such as Sp100 or ISG20 are inducible by type I IFNs (Gongora et al., 1997
; Grotzinger et al., 1996
; Guldner et al., 1992
; Lavau et al., 1995
; Nason-Burchenal et al., 1996
; Regad & Chelbi-Alix, 2001
) and it has been reported that PML itself has antiviral activity (Chee et al., 2003
; Chelbi-Alix et al., 1998
; Djavani et al., 2001
; Regad et al., 2001
). As a countermeasure, certain viruses seem to alter the composition and possibly the function of PML NBs. Thus, a number of DNA viruses associate with PML NBs and change their morphology (Everett, 2001
). Less is known about possible interactions between RNA viruses and PML NBs. Infection with lymphocytic choriomeningitis virus leads to the relocation of the PML protein from the nucleus to the cytoplasm (Borden et al., 1998
). Rabies virus expresses two proteins that interact with the PML protein and alter its distribution (Blondel et al., 2002
). Interestingly, both these RNA viruses replicate in the cytoplasm but have the capacity to alter the composition of PML NBs in the nucleus. The exact role of these virushost interactions is still unclear.
In the present study, we investigated the role of PML NBs in the nuclear localization of Mx1 and its antiviral activity against influenza A virus. We found that cells lacking PML NBs supported influenza virus growth to the same degree as wild-type cells, indicating that PML NBs are not involved in virus multiplication. Moreover, the intranuclear localization of Mx1 was unchanged in these cells. Likewise, nuclear Mx proteins of different species co-localized with murine Mx1 when co-expressed, indicating that Mx proteins prefer particular subnuclear domains, which may constitute specific Mx nuclear domains. Finally, the antiviral activity of Mx1 against influenza A virus was unchanged in cells lacking PML NBs.
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METHODS |
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Antibodies.
Monoclonal anti-MxA antibody M143 recognizes a conserved N-terminal epitope and shows broad cross-reactivity to Mx proteins from many species, including mouse Mx1 (Flohr et al., 1999). Polyclonal rabbit anti-Mx1 antibody is a hyperimmune serum of rabbits immunized with highly purified recombinant Mx1 protein produced in E. coli. Polyclonal anti-influenza A virus hyperimmune rabbit serum has been described previously (Haller et al., 1976
). Polyclonal rabbit antiserum against Daxx (M-112), polyclonal rabbit (H-238) and goat (A-20) antisera against PML and polyclonal rabbit antiserum against the myc epitope (A-14) were obtained from Santa Cruz Biotechnologies. Anti-FLAG tag (M2) and anti-SC35 monoclonal antibodies were from Sigma-Aldrich. Monoclonal mouse (5P10-p) and polyclonal rabbit anti-p80 coilin antibodies were kind gifts from Werner Franke (DKFZ, Heidelberg, Germany) and Angus Lamond (Wellcome Trust Biocentre, University of Dundee, Dundee, UK), respectively. Secondary antibodies used were donkey anti-mouse IgG, donkey anti-rabbit IgG and donkey anti-goat IgG conjugated to Cy2, Cy3 or Cy5, as required (Jackson ImmunoResearch Laboratories).
Plasmids.
Plasmids pHMG-Mx1 (Kolb et al., 1992), pHMG-Mx1(K49A) (Pitossi et al., 1993
), pHMG-Mx1(K49M) (Pitossi et al., 1993
) and pHMG-FLAG-MxA (Ponten et al., 1997
) have been described previously. Plasmid pHMG-FlagMx1 was kindly provided by Georg Kochs (Department of Virology, University of Freiburg, Freiburg, Germany). Briefly, the FLAG sense oligonucleotide (5'-CGGTACCGAAGATGGACTACAAGGACGACGATGA-3') flanked by ClaI adaptors and the complementary oligonucleotide were inserted into the ClaI restriction site of pSP65-Mx1(ClaI) (Zurcher et al., 1992b
); the resulting FLAGMx1 cDNA was transferred to plasmid pHMG (Gautier et al., 1989
). The resulting construct pHMG-FlagMx1 carried the FLAG sequence as a 5'-terminal extension of the Mx1 open reading frame. Plasmid pHMG-rat Mx1 was produced by inserting the rat Mx1 cDNA (Meier et al., 1988
) into plasmid pHMG (Gautier et al., 1989
). pEGFP-Mx1 was kindly provided by Jovan Pavlovic (Institute for Medical Virology, University of Zürich, Zürich, Switzerland), pEGFP-PML was a kind gift from Hans Will (Heinrich-Pette-Institut, Hamburg, Germany). Plasmid pCImycTMxA was generated by replacing the BamHINotI fragment of plasmid pCImycPKM-C1 (Engelhardt et al., 2003
) with a PCR product containing the open reading frame of TMxA (Zurcher et al., 1992a
). Retroviral vector plasmids pRV-Mx1, pRV-Mx1(K49A) and pRV-Mx1(K49M) were generated by inserting PCR products containing the open reading frames of the respective proteins into vector pRRL-sin.hCMV.MCS.pre.DEco via the BamHI and EcoRV sites. Plasmids pRRL-sin.hCMV.MCS.pre.DEco, pRSV-Rev, pMDLg/pRRE and pHCMV-VSV-G for the generation of lentiviral vectors have been described previously (Dull et al., 1998
; Zufferey et al., 1998
) (kind gifts from Hans Will).
Transfections and transductions.
Cells were seeded into six-well plates or 24-well plates (with or without glass coverslips) and transfected the following day using Lipofectamine (Life Technologies) according to the manufacturer's instructions.
Retroviral vector-containing supernatants were produced in 293T cells. 293T cells in 90 mm dishes were transfected with plasmids pRSV-Rev, pMDLg/pRRE, pHCMV-VSV-G and pRV-Mx1, pRV-Mx1(K49A) or pRV-Mx1(K49M) using Metafectene (Biontex) according to the manufacturer's instructions. Supernatants were harvested at different time points between 15 and 52 h post-transfection and titrated by transduction of wild-type MEFs and immunofluorescence analysis using anti-Mx antibody M143.
For transduction with retroviral vectors, cells were seeded into 24-well plates, washed with PBS the next day and incubated with retroviral vector-containing supernatants for 46 h in the presence of 8 µg polybrene ml1 (Sigma-Aldrich). More virus-containing supernatant was added and cells were incubated at 37 °C overnight. The inoculum was removed and replaced with DMEM plus 20 % FCS. Cells were processed for immunofluorescence staining or infection by influenza A virus (see below) 36 days post-transduction.
Antiviral test.
PML/ or control wild-type MEFs were seeded into the wells of a 24-well plate and transduced the next day with retroviral vectors expressing Mx1, Mx1(K49A) or Mx1(K49M). The cells were split 3 days later into a 24-well plate containing glass coverslips, infected the following day with influenza A virus M-TUR and fixed 6 h post-infection in 3 % paraformaldehyde. Immunofluorescence staining was done as described below with a polyclonal rabbit anti-influenza A virus serum and monoclonal anti-Mx antibody M143. For quantification, cells expressing Mx1 or mutant Mx1 proteins and influenza A virus antigens and cells expressing only Mx1 proteins without influenza A virus antigens were counted to determine the infection rate of Mx-expressing cells. The infection rate of cells not expressing Mx1 proteins on the same coverslips was determined in parallel.
Immunofluorescence staining and confocal microscopy.
Immunofluorescence staining was carried out as described previously (Engelhardt et al., 2001). In some experiments, DNA was counterstained by incubation in Bisbenzimide (0·33 mg ml1 in methanol; Sigma-Aldrich) for 10 min or by incubation in TO-PRO-3 iodide (diluted 1 : 1000; Molecular Probes) for 30 min. Coverslips were mounted in Mowiol (Sigma-Aldrich) containing DABCO [1,4-diazabicyclo(2.2.2)octane; Sigma-Aldrich].
Analysis was done on a Leica DM-IRBE microscope using a 63x objective and appropriate filter sets (Leica Microsystems). Confocal microscopy was performed on a Leica TCS SP2 attached to a Leica DM-IRBE microscope with a 63x objective and on a Radiance 2000 confocal microscope (Bio-Rad) attached to a Nikon Eclipse TE300 microscope with a 60x objective. Image acquisition was done using the appropriate proprietary confocal software, recording the different channels separately. Figures were assembled using Adobe Photoshop, Adobe Illustrator and MetaMorph (Universal Imaging Corporation).
Distance measurements and statistical analysis.
Confocal images of single planes were processed separately for each channel using MetaMorph software. Images were smoothed by applying a median filter, followed, where necessary, by conversion to binary images, pixel erosion and segmentation. Finally, the coordinates of the centroids of the individual domains were exported to Microsoft Excel. For each centroid of PML, SC35 or coilin, the distance to the nearest Mx1 dot was calculated. At least 30 nuclei for each set of labellings were used for statistical analysis. Mean minimum distances, calculated as the mean of all minimum distances determined in one experiment, were compared using Student's t-test. In the case of coilinMx1 measurements, the data were further split. The mean of all per-cell means of the minimum PMLMx1 distances was used as an arbitrary threshold value to classify the degree of association between PML and Mx1; cells with a per-cell mean minimum distance below this threshold were termed high PMLMx1 association, and all others low PMLMx1 association. Means of minimum coilinMx1 distances were determined for each of these two groups and compared using Student's t-test. Differences were considered statistically significant for P<0·01.
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RESULTS |
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Mx1 protein has antiviral activity in the absence of PML NBs
We assessed whether PML NBs were required for the antiviral activity of Mx1. Wild-type and PML/ cells were transduced with lentiviral vectors expressing Mx1 and were subsequently infected with influenza A virus. The cells were fixed 56 h after infection and processed for immunofluorescence analysis, using specific antibodies against Mx1 and viral proteins. The infection rate was quantified as indicated in Methods. As controls, two antivirally inactive mutant forms of Mx1, namely Mx1(K49M) and Mx1(K49A) (Pitossi et al., 1993), were expressed and analysed in parallel. Fig. 3
(a) shows that Mx1 protected both wild-type and PML/ cells from infection. As expected, Mx1(K49A) had no antiviral effect in either cell culture. Fig. 3(b)
shows the quantification of a representative experiment. Mx1 inhibited infection of PML-negative cells to the same degree as wild-type cells, while the mutant proteins Mx1(K49M) and Mx1(K49A) had no effect. Clearly, Mx1 inhibited influenza A virus in the absence of PML, indicating that its antiviral function was not dependent on PML NBs.
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We also analysed the relationship of Mx1 dots with other prominent nuclear structures, namely SC35 speckles and Cajal bodies. Vero cells transfected with the plasmid expressing GFPMx1 were labelled with anti-PML and anti-SC35 or with anti-PML and anti-p80 coilin antibodies and examined by confocal microscopy. As expected, PML and GFPMx1 were frequently found closely associated (Fig. 6ad). It should be noted that Vero cells without IFN treatment have a fairly small number of PML NBs per nucleus. SC35 speckles were often separated from Mx1 dots; however, a number of SC35 speckles displayed association with Mx1 dots and/or PML NBs (Fig. 6a and b
and enlargements a1b2). Similarly, Cajal bodies, as revealed by labelling p80 coilin, were sometimes in close proximity to PML NBs as well as to Mx1 dots, whereas in other instances they occupied distinct areas of the nucleus (Fig. 6c, d
). This varied pattern of localization and association prompted us to analyse the distances between these nuclear structures statistically, similar to previously reported approaches (Shiels et al., 2001
; Wang et al., 2004
). The distance between each PML, SC35 or coilin domain and their nearest Mx1 dot was determined as described in Methods. The mean minimum SC35Mx1 distance was larger than the mean minimum PMLMx1 distance (Fig. 6e
), confirming our qualitative observation that Mx1 dots were associated more closely with PML NBs than with SC35 speckles. In contrast, the mean minimum distance between coilin and Mx1 dots did not differ significantly from the mean PMLMx1 distance. Since the association of Mx1 with PML NBs depends, at least in part, on expression levels of Mx1 and its accumulation in bigger dots (see, for example, Fig. 2ac
), we divided the distance data for Cajal bodies into two groups, nuclei with low and high association between PML and Mx1 (see Methods for details). Comparison of the mean minimum coilinMx1 distances between these two groups revealed that the association of Cajal bodies with Mx1 dots reflected the degree of association between PML NBs and Mx1 dots, in as far as the mean minimum coilinMx1 distance was significantly lower in cells considered high PMLMx1 association.
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DISCUSSION |
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It should be noted that the association of PML NBs and Mx1 is rarely complete. In most cells, only a subset of Mx1 dots is juxtaposed to or partially overlapping with PML NBs. Furthermore, cells expressing low levels of Mx1 often show a fine-punctate distribution of Mx1 with little or no obvious association with PML NBs, suggesting that expression levels determine localization. Thus, in IFN-treated cells, Mx1 may first accumulate in the form of small aggregates that fuse and grow bigger when protein levels increase. Larger aggregates may then form at particular sites in the nucleus that are found frequently in the immediate vicinity of PML NBs.
In cells lacking PML NBs, the sites of Mx1 dot formation seem to be preserved. It remains to be seen whether Mx1 interacts with other proteins in these areas. Proteinprotein interactions in PML NBs and probably other subnuclear domains are highly dynamic. Daxx, Sp100, SUMO-1, TOPORS, FLASH, PIAS-1 and PKM/HIPK-2 are partially associated with a subset of Mx1 domains and may be engaged in such dynamic Mx1 interactions (Engelhardt et al., 2001; Trost et al., 2000
; this study and data not shown).
An intriguing observation was the finding that nuclear Mx proteins of three different species exhibited an almost identical subnuclear distribution. The nuclear Mx1 protein of the rat behaved like mouse Mx1 and displayed dots that were frequently associated with PML NBs. Surprisingly, the human MxA protein, known to reside in the cytoplasm, adopted an Mx1-like localization upon artificial translocation into the cell nucleus. This nuclear form of MxA co-localized with mouse Mx1 in PML-associated dots and tracks. This co-localization was unexpected because the two proteins do not interact, as shown in an in vivo assay. Obviously, nuclear Mx proteins accumulate in their own distinct nuclear domains, which we have tentatively called Mx nuclear domains. These domains are functionally independent of PML NBs, albeit often closely associated with PML NBs in wild-type cells. We propose that the same proteinprotein interactions leading to dot formation of mouse Mx1 in the vicinity of PML NBs also apply to other nuclear Mx proteins, including the nuclear form of MxA.
We observed that Mx nuclear domains' were not exclusively associated with PML NBs, but could also be found in the vicinity of Cajal bodies and, to a lesser degree, of SC35 speckles. Association of one nuclear domain with more than one type of other nuclear structure has been reported in the literature (Wang et al., 2002) and may be a consequence of the highly dynamic nature of most nuclear structures or of the multitude of proteinprotein interactions of which some proteins are capable. The putative interaction partners of Mx1 are mostly components of PML NBs, suggesting a biochemical link of Mx1 to PML NBs. However, it remains possible that proteins will be found that link Mx1 to other nuclear structures. It is noteworthy that, in the natural situation of virus infection, Mx1 is induced by IFN, which also leads to an increase in the number of PML NBs but not of SC35 speckles and Cajal bodies, resulting in the high degree of association seen in mouse cells upon IFN treatment (Engelhardt et al., 2001
).
An interesting question is whether dot formation is essential for Mx1 activity. It is possible that nuclear Mx proteins require the dot-forming domains in order to exert their antiviral activity against influenza viruses and other orthomyxoviruses. The sites of virus transcription and replication in the nucleus are presently not known. It is conceivable that these viral processes occur at or near the Mx nuclear domains' described here. Alternatively, Mx1 dots may serve as storage depots for excess Mx1 protein. This would imply that active Mx1 is recruited from these depots to sites of virus transcription and replication. A similar depot model has also been proposed for PML NBs (Negorev & Maul, 2001). If the sole purpose of Mx nuclear domains is to serve as storage depots, these domains are expected to be dispensable for antiviral activity. This is in line with previous findings showing that Mx1(C71S), a mutant form of Mx1 with a single amino acid exchange at position 71, is antiviral, although this mutant does not form the characteristic nuclear dots (Toyoda et al., 1995
). However, using our high-expression vectors, this Mx1 mutant was able to accumulate in nuclear dots in a majority of cells (see Supplementary Figure in JGV Online), suggesting that dot formation most likely depends on protein expression levels, as discussed above. All other mutants of Mx1 with a diffuse intranuclear distribution are reported not to have antiviral activity (Garber et al., 1993
). On the other hand, some inactive variants of Mx1 accumulated in dots in the nucleus (Pitossi et al., 1993
) (see also Fig. 3a
). Therefore, it is presently not possible to decide whether or not Mx1 dots play a direct role in the antiviral activity of Mx1. Further mutational studies will be needed to clarify this issue.
Our present study does not support the view that PML itself has antiviral activity against influenza viruses (Chelbi-Alix et al., 1998). In our hands, both wild-type and PML/ cells were equally permissive for the influenza A virus strain used. This also suggests that PML NBs do not play an essential role in the influenza virus life cycle. In contrast, Mx1 was clearly antiviral, even in the absence of PML. Mx GTPases are members of the dynamin superfamily of large GTPases and may have specialized cellular functions in addition to their antiviral activity (Haller & Kochs, 2002
). It will be interesting to define further the significance of the Mx nuclear domains' described here for the cellular and antiviral functions of this class of nuclear GTPases.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Blondel, D., Regad, T., Poisson, N., Pavie, B., Harper, F., Pandolfi, P. P., de The, H. & Chelbi-Alix, M. K. (2002). Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies. Oncogene 21, 79577970.[CrossRef][Medline]
Borden, K. L. (2002). Pondering the promyelocytic leukemia protein (PML) puzzle: possible functions for PML nuclear bodies. Mol Cell Biol 22, 52595269.
Borden, K. L., Campbell Dwyer, E. J. & Salvato, M. S. (1998). An arenavirus RING (zinc-binding) protein binds the oncoprotein promyelocyte leukemia protein (PML) and relocates PML nuclear bodies to the cytoplasm. J Virol 72, 758766.
Chee, A. V., Lopez, P., Pandolfi, P. P. & Roizman, B. (2003). Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects. J Virol 77, 71017105.
Chelbi-Alix, M. K., Pelicano, L., Quignon, F., Koken, M. H., Venturini, L., Stadler, M., Pavlovic, J., Degos, L. & de The, H. (1995). Induction of the PML protein by interferons in normal and APL cells. Leukemia 9, 20272033.[Medline]
Chelbi-Alix, M. K., Quignon, F., Pelicano, L., Koken, M. H. & de The, H. (1998). Resistance to virus infection conferred by the interferon-induced promyelocytic leukemia protein. J Virol 72, 10431051.
Der, S. D., Zhou, A., Williams, B. R. & Silverman, R. H. (1998). Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci U S A 95, 1562315628.
Djavani, M., Rodas, J., Lukashevich, I. S., Horejsh, D., Pandolfi, P. P., Borden, K. L. & Salvato, M. S. (2001). Role of the promyelocytic leukemia protein PML in the interferon sensitivity of lymphocytic choriomeningitis virus. J Virol 75, 62046208.
Dreiding, P., Staeheli, P. & Haller, O. (1985). Interferon-induced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses. Virology 140, 192196.[Medline]
Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D. & Naldini, L. (1998). A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 84638471.
Engelhardt, O. G., Ullrich, E., Kochs, G. & Haller, O. (2001). Interferon-induced antiviral Mx1 GTPase is associated with components of the SUMO-1 system and promyelocytic leukemia protein nuclear bodies. Exp Cell Res 271, 286295.[CrossRef][Medline]
Engelhardt, O. G., Boutell, C., Orr, A., Ullrich, E., Haller, O. & Everett, R. D. (2003). The homeodomain-interacting kinase PKM (HIPK-2) modifies ND10 through both its kinase domain and a SUMO-1 interaction motif and alters the posttranslational modification of PML. Exp Cell Res 283, 3650.[CrossRef][Medline]
Everett, R. D. (2001). DNA viruses and viral proteins that interact with PML nuclear bodies. Oncogene 20, 72667273.[CrossRef][Medline]
Flohr, F., Schneider-Schaulies, S., Haller, O. & Kochs, G. (1999). The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures. FEBS Lett 463, 2428.[CrossRef][Medline]
Garber, E. A., Hreniuk, D. L., Scheidel, L. M. & van der Ploeg, L. H. (1993). Mutations in murine Mx1: effects on localization and antiviral activity. Virology 194, 715723.[CrossRef][Medline]
Gautier, C., Mehtali, M. & Lathe, R. (1989). A ubiquitous mammalian expression vector, pHMG, based on a housekeeping gene promoter. Nucleic Acids Res 17, 8389.[Medline]
Gongora, C., David, G., Pintard, L., Tissot, C., Hua, T. D., Dejean, A. & Mechti, N. (1997). Molecular cloning of a new interferon-induced PML nuclear body-associated protein. J Biol Chem 272, 1945719463.
Grotzinger, T., Sternsdorf, T., Jensen, K. & Will, H. (1996). Interferon-modulated expression of genes encoding the nuclear-dot-associated proteins Sp100 and promyelocytic leukemia protein (PML). Eur J Biochem 238, 554560.[Abstract]
Guldner, H. H., Szostecki, C., Grotzinger, T. & Will, H. (1992). IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis. J Immunol 149, 40674073.
Haller, O. & Kochs, G. (2002). Interferon-induced Mx proteins: dynamin-like GTPases with antiviral activity. Traffic 3, 710717.[CrossRef][Medline]
Haller, O., Arnheiter, H. & Lindenmann, J. (1976). Genetically determined resistance to infection by hepatotropic influenza A virus in mice: effect of immunosuppression. Infect Immun 13, 844854.[Medline]
Haller, O., Frese, M., Rost, D., Nuttall, P. A. & Kochs, G. (1995). Tick-borne thogoto virus infection in mice is inhibited by the orthomyxovirus resistance gene product Mx1. J Virol 69, 25962601.[Abstract]
Ishov, A. M., Sotnikov, A. G., Negorev, D., Vladimirova, O. V., Neff, N., Kamitani, T., Yeh, E. T., Strauss, J. F., III & Maul, G. G. (1999). PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1. J Cell Biol 147, 221234.
Israel, A. (1979). Preliminary characterization of the particles from productive and abortive infections of L cells by fowl plague virus. Ann Microbiol (Paris) 130B, 85100.[Medline]
Kochs, G., Trost, M., Janzen, C. & Haller, O. (1998). MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures. Methods 15, 255263.[CrossRef][Medline]
Kochs, G., Haener, M., Aebi, U. & Haller, O. (2002). Self-assembly of human MxA GTPase into highly ordered dynamin-like oligomers. J Biol Chem 277, 1417214176.
Kolb, E., Laine, E., Strehler, D. & Staeheli, P. (1992). Resistance to influenza virus infection of Mx transgenic mice expressing Mx protein under the control of two constitutive promoters. J Virol 66, 17091716.[Abstract]
Lavau, C., Marchio, A., Fagioli, M. & 7 other authors (1995). The acute promyelocytic leukaemia-associated PML gene is induced by interferon. Oncogene 11, 871876.[Medline]
Lindenmann, J., Deuel, E., Fanconi, S. & Haller, O. (1978). Inborn resistance of mice to myxoviruses: macrophages express phenotype in vitro. J Exp Med 147, 531540.[Abstract]
Meier, E., Fah, J., Grob, M. S., End, R., Staeheli, P. & Haller, O. (1988). A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells. J Virol 62, 23862393.[Medline]
Melen, K. & Julkunen, I. (1997). Nuclear cotransport mechanism of cytoplasmic human MxB protein. J Biol Chem 272, 3235332359.
Melen, K., Ronni, T., Broni, B., Krug, R. M., von Bonsdorff, C. H. & Julkunen, I. (1992). Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper. J Biol Chem 267, 2589825907.
Nakayama, M., Yazaki, K., Kusano, A., Nagata, K., Hanai, N. & Ishihama, A. (1993). Structure of mouse Mx1 protein. Molecular assembly and GTP-dependent conformational change. J Biol Chem 268, 1503315038.
Nason-Burchenal, K., Gandini, D., Botto, M., Allopenna, J., Seale, J. R., Cross, N. C., Goldman, J. M., Dmitrovsky, E. & Pandolfi, P. P. (1996). Interferon augments PML and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line. Blood 88, 39263936.
Negorev, D. & Maul, G. G. (2001). Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot. Oncogene 20, 72347242.[CrossRef][Medline]
Piazza, F., Gurrieri, C. & Pandolfi, P. P. (2001). The theory of APL. Oncogene 20, 72167222.[CrossRef][Medline]
Pitossi, F., Blank, A., Schroder, A., Schwarz, A., Hussi, P., Schwemmle, M., Pavlovic, J. & Staeheli, P. (1993). A functional GTP-binding motif is necessary for antiviral activity of Mx proteins. J Virol 67, 67266732.[Abstract]
Ponten, A., Sick, C., Weeber, M., Haller, O. & Kochs, G. (1997). Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity. J Virol 71, 25912599.[Abstract]
Rasheed, Z. A., Saleem, A., Ravee, Y., Pandolfi, P. P. & Rubin, E. H. (2002). The topoisomerase I-binding RING protein, topors, is associated with promyelocytic leukemia nuclear bodies. Exp Cell Res 277, 152160.[CrossRef][Medline]
Regad, T. & Chelbi-Alix, M. K. (2001). Role and fate of PML nuclear bodies in response to interferon and viral infections. Oncogene 20, 72747286.[CrossRef][Medline]
Regad, T., Saib, A., Lallemand-Breitenbach, V., Pandolfi, P. P., de The, H. & Chelbi-Alix, M. K. (2001). PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator. EMBO J 20, 34953505.
Salomoni, P. & Pandolfi, P. P. (2002). The role of PML in tumor suppression. Cell 108, 165170.[Medline]
Shiels, C., Islam, S. A., Vatcheva, R., Sasieni, P., Sternberg, M. J., Freemont, P. S. & Sheer, D. (2001). PML bodies associate specifically with the MHC gene cluster in interphase nuclei. J Cell Sci 114, 37053716.[Medline]
Staeheli, P. & Haller, O. (1985). Interferon-induced human protein with homology to protein Mx of influenza virus-resistant mice. Mol Cell Biol 5, 21502153.[Medline]
Staeheli, P., Haller, O., Boll, W., Lindenmann, J. & Weissmann, C. (1986). Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44, 147158.[Medline]
Staeheli, P., Grob, R., Meier, E., Sutcliffe, J. G. & Haller, O. (1988). Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol Cell Biol 8, 45184523.[Medline]
Toyoda, T., Asano, Y. & Ishihama, A. (1995). Role of GTPase activity of murine Mx1 protein in nuclear localization and anti-influenza virus activity. J Gen Virol 76, 18671869.[Abstract]
Trost, M., Kochs, G. & Haller, O. (2000). Characterization of a novel serine/threonine kinase associated with nuclear bodies. J Biol Chem 275, 73737377.
Wang, I. F., Reddy, N. M. & Shen, C. K. (2002). Higher order arrangement of the eukaryotic nuclear bodies. Proc Natl Acad Sci U S A 99, 1358313588.
Wang, J., Shiels, C., Sasieni, P., Wu, P. J., Islam, S. A., Freemont, P. S. & Sheer, D. (2004). Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions. J Cell Biol 164, 515526.
Zhong, S., Muller, S., Ronchetti, S., Freemont, P. S., Dejean, A. & Pandolfi, P. P. (2000a). Role of SUMO-1-modified PML in nuclear body formation. Blood 95, 27482752.
Zhong, S., Salomoni, P. & Pandolfi, P. P. (2000b). The transcriptional role of PML and the nuclear body. Nat Cell Biol 2, E85E90.[CrossRef][Medline]
Zufferey, R., Dull, T., Mandel, R. J., Bukovsky, A., Quiroz, D., Naldini, L. & Trono, D. (1998). Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72, 98739880.
Zurcher, T., Pavlovic, J. & Staeheli, P. (1992a). Mechanism of human MxA protein action: variants with changed antiviral properties. EMBO J 11, 16571661.[Abstract]
Zurcher, T., Pavlovic, J. & Staeheli, P. (1992b). Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus. J Virol 66, 50595066.[Abstract]
Received 11 November 2003;
accepted 20 April 2004.
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