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Assay and Functional Analysis of Dynamin‐Like Mx Proteins
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[55] Assay and Functional Analysis of Dynamin‐Like Mx Proteins By GEORG KOCHS, MIKE REICHELT, DGANIT DANINO, JENNY E. HINSHAW , and OTTO HALLER Abstract
Mx proteins are interferon‐induced large guanosin triphosphatases (GTPases) that share structural and functional properties with dynamin and dynamin‐like proteins, such as self‐assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a 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 association of the human MxA GTPase with membranes of the endoplasmic reticulum seems to support its antiviral function by providing an interaction platform that facilitates viral target recognition, MxA oligomerization, and missorting of the resulting multiprotein complex into large intracellular aggregates. Introduction
Mx proteins are members of the superfamily of high molecular weight GTPases (Haller and Kochs, 2002). Many of these dynamin‐like GTPases localize to intracellular membranes and are involved in intracellular trafficking, membrane remodeling, and fission processes (Danino and Hinshaw, 2001; McNiven et al., 2000). Some family members appear to have a different role, in being key components of the early innate immune response against a wide variety of invading pathogens (Haller and Kochs, 2002; Praefcke and McMahon, 2004; Taylor et al., 2004). Accordingly, these GTPases are upregulated by type I ( /b) or type II ( ) interferons (IFN), and they function as intracellular resistance factors capable of restricting the growth of distinct pathogens. The Mx GTPases are expressed exclusively in IFN‐ /b–treated cells (Haller and Kochs, 2002). Human MxA, a 78 kDa protein, accumulates in the cytoplasm of IFN‐ treated cells and inhibits the replication of a wide range of viruses, including influenza viruses, measles virus, and bunyaviruses (Haller and Kochs, 2002). Biochemical and cell culture studies suggest that MxA interacts
METHODS IN ENZYMOLOGY, VOL. 404 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)04055-3
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directly with viral target structures. MxA was shown to bind to the nucleocapsids of Thogoto virus (THOV), an influenza‐like orthomyxovirus. As a consequence, the THOV nucleocapsids were prevented from entering the nucleus where transcription and replication of the viral genome normally occurs (Kochs and Haller, 1999b; Weber et al., 2000). MxA also inhibits the replication of La Crosse virus (LACV), a bunyavirus with a cytoplasmic replication phase. In this case, MxA binds to the viral nucleocapsid (N) protein and forms large copolymers that accumulate in the perinuclear area (Kochs et al., 2002b). Overall, MxA seems to act by interfering with the proper transport of critical viral components to their ultimate target compartments in infected cells. Membrane association and homo‐oligomerization are essential for the biological function of dynamin‐like proteins (Danino and Hinshaw, 2001; Praefcke and McMahon, 2004). MxA forms homo‐oligomers both in vitro and in vivo (Accola et al., 2002; Kochs et al., 2002a). Three distinct domains are involved in the self‐assembly process: (i) a ‘‘self‐assembly sequence’’ (SAS) that is located within the N‐terminal G‐domain and is conserved in all members of the dynamin‐like GTPases (Nakayama et al., 1993); (ii) a central interactive domain (CID) that mediates the association with the C‐terminal part of the molecule (Ponten et al., 1997); and (iii) a leucine zipper motif (LZ) at the extreme C‐terminus that interacts with the CID (Schumacher and Staeheli, 1998). The interaction between LZ and CID results in increased GTPase activity, indicating that the LZ region acts as a ‘‘GTPase effector domain’’ (GED) (Schwemmle et al., 1995), similar to the GED of dynamin. Furthermore, intra‐ and intermolecular interactions are critical for protein stability and recognition of viral target structures (Flohr et al., 1999; Janzen et al., 2000; Schwemmle et al., 1995). MxA protein localizes to a subcompartment of the smooth endoplasmic reticulum, suggesting that membrane binding and compartmentalization are important for its antiviral function (Accola et al., 2002; Reichelt et al., 2004). Our current model proposes that, in IFN‐treated cells, MxA forms large membrane‐associated self‐assemblies that serve as a stable storage pool from which MxA monomers are transiently released. The equilibrium between assembled and monomeric forms is presumably regulated by the GTPase activity. Upon infection, MxA monomers sense viral target structures and, by binding to them, form new assemblies involving specific viral components. Depending on the local concentration of the binding partners, more and more MxA molecules are recruited into these membrane‐associated copolymers, leading to mislocalization of the viral components and to viral inhibition (Haller and Kochs, 2002).
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Methods
Expression and Purification of MxA Recombinant human MxA is expressed in E. coli as an N‐terminal histidine‐tagged protein using the pQE‐9 vector system (Qiagen, Hilden, Germany), as described (Kochs et al., 2002a; Richter et al., 1995). E. coli strain M15 carrying the pQE‐9‐MxA expression plasmid is incubated at 28 in LB (Luria‐Bertani) medium containing 100 g/ml ampicillin and 25 g/ml kanamycin. At an optical density of 0.3 at 600 nm, MxA expression is induced by adding 0.03 mM isopropyl‐b‐D‐ thiogalactopyranoside followed by further incubation for 2 h. The cells are harvested at an optical density of 0.6 to 0.8 by centrifugation. The bacterial pellet can be stored at 70 . For lysis, the pellet from a 1 l culture is resuspended in 10 ml of buffer A (50 mM Tris, pH 8.0, 500 mM NaCl, 5 mM MgCl2, 0.1% NP40, 10% glycerol, 2 mM imidazole, and 7 mM b‐mercaptoethanol). Lysis is performed on ice by seven cycles of sonication for 30 s each with a Branson B15 sonifier. The homogenate is clarified by centrifugation for 25 min at 20,000g. The resulting supernatant (Fig. 1A and B, lanes 1) is incubated with 800 l Ni‐nitrilotriacetic acid‐ NTA agarose (Qiagen) by end‐over‐end rotation for 2 h at 4 . Then the agarose beads are poured into a column of 5 mm in diameter and washed with 30 ml of buffer A containing 30 mM imidazole followed by a 10 ml washing step with buffer B (like buffer A but with 100 mM NaCl and 2 mM imidazole). His‐MxA is eluted in 0.5 ml steps with buffer B containing 250 mM imidazole (Fig. 1A and B, lanes 2). For further purification and concentration, the Ni‐eluate is directly loaded on a Mono Q anion exchange column (1 ml, 5 50 mm; Amersham‐Pharmacia, Freiburg, Germany) equilibrated in buffer C (50 mM Tris, pH 8.0, 2 mM MgCl2, 100 mM NaCl, 10% glycerol, 0.05% NP40, 1.4 mM b‐mercaptoethanol). After washing with 5 ml of buffer C, His‐MxA is eluted with a linear gradient of 5 ml from 0.1 to 1 M NaCl in buffer C. His‐MxA typically elutes between 300 to 500 mM NaCl (Fig. 1A and B, lanes 3) with a protein concentration of about 1 mg/ml. The purified protein can be stored at 70 . The GTPase activity of MxA (the hydrolysis of GTP into GDP and inorganic phosphate) is measured as described (Richter et al., 1995). A GTPase inactive mutant with an amino acid exchange in the N‐terminal G‐domain, MxA(T103A) (Ponten et al., 1997), is used as a negative control. About 1 g of each purified protein is incubated in 50 l of buffer D (50 mM Tris, pH 8.0, 5 mM MgCl2, 100 mM KCl, 10% glycerol, 0.1 mM dithiothreitol (DTT)) with 1 mM GTP and [32P]‐ ‐GTP (200 nCi) in the
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FIG. 1. Purification and GTPase activity of MxA. (A) Coomassie blue‐stained SDS‐ polyacrylamide gel loaded with fractions obtained during the purification of His‐MxA expressed in E. coli. Lane 1, supernatant after centrifugation; lane 2, eluate from the Ni‐NTA affinity column; lane 3, eluate from the Mono Q ion exchange column. (B) Western blot analysis. One‐fifth of the fractions shown in (A) were blotted on a PVDF membrane and analyzed using the monoclonal anti‐MxA antibody M143 (dilution 1:500) (Flohr et al., 1999). (C) GTPase activity of recombinant wild‐type MxA and MxA(T103A), a GTPase‐ inactive mutant (Ponten et al., 1997). Thin‐layer chromatogram of GTP and GDP after incubation with Mono Q‐purified wild‐type MxA or MxA(T103A) (1 mg) for 0, 30, and 60 min at 37 .
presence of 100 nM AMP‐PNP. At various time points the reaction is terminated by mixing 10 l of the reaction mixture with the same volume of 2 mM ethylenediaminetetraacetic acid (EDTA) in 0.5% sodium dodecyl sulfate (SDS). To separate the radio‐labeled GDP product from the GTP substrate, 1.5 l of the stopped reaction is spotted onto a polyethyleneimine‐cellulose thin‐layer chromatography plate (PEI‐5725, Merck, Darmstadt, Germany) and developed in 1 M LiCl and 1 M acetic acid. Figure 1C
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shows an autoradiography of a dried plate. The result demonstrates that Mx proteins, in contrast to small Ras‐like GTPases, exhibit a high intrinsic GTPase activity. Oligomerization of MxA MxA Oligomerization in Living Cells Mx‐proteins form high molecular weight oligomers in vivo and in vitro as shown by chemical cross‐linking, gel filtration, and ultrastructural analysis of recombinant proteins (Kochs et al., 2002a; Melen et al., 1992; Richter et al., 1995). To monitor their self‐assembly in living cells, we established a nuclear translocation assay (Ponten et al., 1997). The idea of this assay is that in case of homooligomerization, an artificial nuclear form of MxA should be able to drag the normally cytoplasmic MxA protein into the nucleus. For this approach, MxA is modified by a foreign nuclear translocation signal (NLS). The NLS of the large T‐antigen of SV40 is fused to the N terminus of MxA resulting in TMxA that translocates into the nucleus when expressed in eukaryotic cells (Zu¨ rcher et al., 1992). To distinguish nuclear TMxA from the normal cytoplasmic MxA, wild‐type MxA is N‐terminally tagged with a FLAG peptide (Hopp, 1988). Mouse 3T3 cells are seeded onto glass coverslips. After 4 h, the cells are transfected with pHMG‐expression plasmids using 2 l of Lipofectamine transfection reagent (Invitrogen, Karlsruhe, Germany). At 20 h post‐transfection the cells are fixed with 3% paraformaldehyde (PFA) in phosphate‐buffered saline (pH 7.4) (PBS), permeabilized with 0.5% Triton X‐100, and stained using the monoclonal anti‐Mx antibody M143 (Flohr et al., 1999) or a monoclonal anti‐FLAG antibody (Sigma) in PBS with 5% fish gelatin. After three washing steps with PBS, the antigen‐bound primary antibodies are detected with fluorophore (Cy2)‐conjugated donkey antibodies (Dianova, Hamburg, Germany) and analyzed with a fluorescence microscope. Figure 2A shows that FLAG‐tagged wild‐type MxA accumulates in cytoplasmic dots (left panel) and TMxA in the nucleus (right panel). Upon coexpression of both cDNAs, the wild‐type MxA, detected by the FLAG antibody, predominantly accumulates in the nucleus, indicating a tight association with TMxA (Fig. 2A, middle panel). Interestingly, a fraction of FLAG‐MxA remains in the cytoplasm, presumably bound to intracellular membranes (see following). This approach might also be suitable to study protein‐protein interactions of other candidate proteins in living cells.
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FIG. 2. MxA oligomerization in vivo and in vitro. (A) TMxA and FLAG‐tagged wild‐type MxA were transfected either separately (left and right) or together (cotransfection, middle panel) in murine 3T3 cells. The subcellular localization of the recombinant proteins was analyzed by indirect immunofluorescence using either the anti‐FLAG or the anti‐MxA antibody M143 (dilution 1:500). (B) Cryo‐TEM images of MxA self‐assemblies. Purified His‐ MxA dialyzed overnight in low salt buffer with 1 mM GMP‐PCP self‐assembled into rings and open arcs (a). (b) Higher magnification of the rings revealed a structure of two parallel sets of electron dense globular domains (double arrowheads). (c) When dialyzed in the presence of GDP/BeF, MxA self‐organized into long, straight, and ordered complexes (arrows). (Bars ¼ 50 nm).
Structural Analysis of MxA Oligomers by Cryo‐transmission Electron Microscopy In vitro, MxA forms ring‐like and helical oligomers, similar to the structures formed by dynamin (Accola et al., 2002; Kochs et al., 2002a). These structures are studied at higher resolution using transmission electron microscopy at cryogenic temperatures (cryo‐TEM). Cryo‐TEM is based on ultra‐rapid thermal fixation of the sample that preserves the structures at their native state (Danino et al., 1997, 2004). Therefore, it is a powerful method, better suited to study Mx oligomer formation than conventional chemical fixation methods like negative staining.
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Specimens for cryo‐TEM are prepared at a controlled temperature of 25 and saturated water atmosphere in a controlled environment vitrification system. An 8 l drop of purified His‐MxA (1 g/l) in buffer E (20 mM Hepes, pH 7.2, 2 mM EGTA, 1 mM MgCl2, 1 mM DTT, 50 mM NaCl) and 1 mM nucleotide is placed on a TEM grid covered with perforated carbon film (Ted Pella, USA) held by a tweezers. The drop is blotted with a filter paper forming a film of 100–250 nm in thickness, and dropped into liquid ethane at its freezing point of 183 . The specimen is then transferred to liquid nitrogen for storage. Specimens are examined in a Tecnai G2 T12 transmission electron microscope operating at 120 kV using a Gatan 626 cryo holder maintained at below 175 . Imaging is done at low dose exposures to minimize radiation damage of the specimens, and images are recorded digitally on a Gatan 791 wide‐angle cooled CCD camera at effective magnifications of up to 50,000 using specific imaging procedures as described (Danino et al., 2001). Ring‐like MxA structures form upon dialysis of MxA in the presence of 1 mM GMP‐PCP, a nonhydrolyzing analogue of GTP (Fig. 2Ba). Higher magnification images reveal that the rings are composed of two parallel sets of electron dense globular domains that are formed most likely by intra‐ and interdomain associations of the MxA molecules (Fig. 2Bb). Likewise, long, ordered MxA assemblies displaying a ladder pattern are induced by dialysis of MxA against buffer E in the presence of GDP/BeF (1 mM GDP, 5 mM NaF, 500 mM BeCl2, in 5% ethylene glycol) for 20 h at 4 (Fig. 2Bc). Binding of GDP/BeF mimics a transition state during GTP hydrolysis (Ahmadian et al., 1997). This indicates that cryo‐TEM is possibly the method of choice to study the structure and the kinetics of assembly of high molecular weight Mx oligomers. Membrane Association of MxA Interestingly, human MxA is a membrane‐associated large GTPase, although it lacks obvious membrane interaction domains comparable to the PH domain of dynamin (Accola et al., 2002; Reichelt et al., 2004). To study the association of MxA with membranes, Vero cells constitutively expressing human MxA (Frese et al., 1995) are analyzed by differential centrifugation. For this, VA3 cells from two 150 cm2 petri dishes are lysed in 1 ml of buffer F (25 mM Hepes, pH 7.5, 2.5 mM MgCl2, 150 mM NaCl, 1 mM DTT) using a dounce‐homogenizer. The homogenate is centrifuged at 1000g (P1) and the resulting supernatant is subjected first to 10,000g (P10) and then to 100,000g centrifugation (P100 and S100). The pellets are resuspended in buffer F and equal amounts of protein are analyzed by Western blotting using the monoclonal anti‐Mx antibody M143
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FIG. 3. MxA is a membrane‐resident protein. (A) Fractionation of MxA‐expressing cells. Vero cells constitutively expressing high levels of recombinant human MxA (VA3) were compared to control cells lacking MxA (VN36) (Frese et al., 1995). Lysates of VA3 cells were centrifuged first at 1000g (P1), then at 10,000g (P10), and the resulting supernatant at 100,000g (P100 and S100). Equivalent amounts of protein were analyzed by Western blotting using the monoclonal anti‐MxA antibody M143. (B) In vitro interaction of purified His‐MxA with liposomes. MxA was incubated with PS‐liposomes for 1 h at 37 in the presence of GTP and then analyzed by cryo‐TEM. MxA assembles on the surface of a lipid vesicle (a). In the presence of lipid tubes MxA forms ring‐like assemblies (b) with a characteristic Y‐like appearance (arrows). Scale bar ¼ 100 nm.
(Fig. 3A). Approximately 30% of the total MxA protein content is sedimented with the microsomal pellet, P100, suggesting that part of MxA is associated with membranes.
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Structural Analysis of MxA‐membrane Association In Vitro To study in vitro association of MxA with membranes, a 10 g/l solution of phosphatidylserine (PS, Avanti Polar Lipids, Alabaster, AL) in chloroform is dried under a stream of nitrogen, kept under vacuum overnight, and resuspended to a final concentration of 2 g/l in buffer E. Liposomes are generated by extruding the lipid solution 15 times through a 1 m polycarbonate membrane (Avanti Polar Lipids). Purified MxA is incubated with the liposomes (final concentrations 0.3 and 0.25 g/l for MxA and PS, respectively) for 1 h at 37 in the presence of 1 mM GTP, and is subsequently prepared for cryo‐TEM as described above. Figure 3Ba shows that MxA associates with the surface of liposomes, indicating that MxA has the potential to directly interact with lipid membranes. In the presence of lipid tubes, sometimes the formation of ring‐like MxA structures can be detected. At high magnification, these assemblies show an Y‐like shape (Fig. 3Bb), reminiscent of the Y‐shaped assemblies of dynamin around lipid tubes (Zhang and Hinshaw, 2001). MxA Interaction with Viral Target Structures According to our hypothesis, the antiviral action of Mx proteins is based on their direct interaction with viral components essential for gene expression and genome replication (Haller and Kochs, 2002; Kochs and Haller, 1999a; Kochs et al., 2002b). To show direct interaction of human MxA with viral structures, we analyze infected cells by immunofluorescence and coimmunoprecipitation. VA3 cells are seeded on glass coverslips and infected with 10 plaque‐ forming units per cell of La Crosse virus (LACV), a MxA‐sensitive bunyavirus (Frese et al., 1996). After 16 h the cells are stained for triple fluorescence analysis as described above, using the monoclonal anti‐MxA antibody M143, a goat polyclonal anti‐Syntaxin17 antiserum, and a polyclonal rabbit antiserum directed against the nucleoprotein (N) of LACV. Syntaxin17 is a marker for a subcompartment of the smooth endoplasmic reticulum (ER) (Steegmaier et al., 2000). The primary antibodies are detected with fluorophore (Cy2, Cy3, and Cy5)‐conjugated donkey secondary antibodies, respectively, and analyzed with a Leica TCSSP2 confocal laser scanning microscope. Colocalization of MxA with Syntaxin17 clearly shows that MxA is a membrane‐associated protein with a distinct subcellular distribution (Fig. 4Aa and b). The colocalization of MxA with the viral N protein indicates a tight association of membrane‐resident MxA with viral nucleocapsids (Fig. 4Aa and c).
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FIG. 4. MxA forms complexes with viral target structures and cellular membranes. (A) Colocalization of MxA/N protein complexes with smooth ER membranes. VA3 cells were infected with LACV for 16 h, fixed as described above, and stained with monoclonal anti‐ MxA antibody (a, green), goat anti‐Syntaxin17 antibody (b, red), and a rabbit antiserum specific for the viral N protein (c, blue). The primary antibodies were detected with fluorophore (Cy2, Cy3, and Cy5)‐conjugated secondary antibodies, respectively. The pictures were recorded using a Leica TCSSP2 confocal laser scanning microscope. Bar ¼ 8 mm. (B) VA3 cells were infected with LACV or left uninfected. Cells were lysed in the presence or absence of GTP‐gS and subjected to immunoprecipitation using the N‐specific antiserum. Bound MxA was detected by Western blotting using the monoclonal anti‐MxA antibody M143 (from Kochs et al., 2002, # 2002).
Coimmunoprecipitation of MxA with Viral N Protein To verify the MxA/N interaction, virus‐infected VA3 cells from one 150 cm2 petri dish are lysed as described above in buffer G (50 mM Tris, pH 7.5, 0.1% Nonidet P‐40, 5 mM MgCl2, 0.5 mM DTT) in the presence or absence of 200 mM guanosine 50 ‐O‐[ ‐thio]‐triphosphate (GTP‐ S), a nonhydrolyzable GTP analogue. The lysates are incubated with 2 l of the polyclonal rabbit anti‐N antiserum coupled to 30 l of protein A‐sepharose beads (Amersham‐Pharmacia) for 2 h at 4 . After washing the beads
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several times in buffer G, bound proteins are eluted by incubation in SDS‐ containing Laemmli sample buffer for 5 min at 95 and analyzed by Western blotting using the monoclonal anti‐MxA antibody M143. The Western blot (Fig. 4B) demonstrates that MxA interacts with the viral N protein only in the presence of GTP‐ S, indicating that MxA is competent for interaction in its GTP‐bound conformation (Kochs et al., 2002b).
References Accola, M. A., Huang, B., Masri, A. A., and McNiven, M. A. (2002). The antiviral dynamin family member, MxA, tubulates lipids and localizes to the smooth endoplasmic reticulum. J. Biol. Chem. 277, 21829–21835. Ahmadian, M. R., Mittal, R., Hall, A., and Wittinghofer, A. (1997). Aluminium fluoride associates with the small guanine nucleotide binding proteins. FEBS Lett. 408, 315–318. Danino, D., Bernheim‐Groswasser, A., and Talmon, Y. (2001). Digital cryogenic transmission electron microscopy: An advanced tool for direct imaging of complex fluids. Colloids and Surfaces A: Physicochem. Engineer. Aspects 183, 113–122. Danino, D., and Hinshaw, J. E. (2001). Dynamin family of mechanoenzymes. Curr. Opinion Cell Biol. 13, 454–460. Danino, D., Kaplun, A., Lindblom, G., Rilfors, L., Oradd, G., Hauksson, J. B., and Talmon, Y. (1997). Cryo‐TEM and NMR studies of a micelle‐forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B. Chem. Phys. Lipids 85, 75–89. Danino, D., Moon, K. H., and Hinshaw, J. E. (2004). Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin. J. Struct. Biol. 147, 259–267. Flohr, F., Schneider‐Schaulies, S., Haller, O., and 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, 24–28. Frese, M., Kochs, G., Feldmann, H., Hertkorn, C., and Haller, O. (1996). Inhibition of Bunyaviruses, Phleboviruses, and Hantaviruses by human MxA protein. J. Virol. 70, 915–923. Frese, M., Kochs, G., Meier‐Dieter, U., Siebler, J., and Haller, O. (1995). Human MxA protein inhibits tick‐borne Thogoto virus but not Dhori virus. J. Virol. 69, 3904–3909. Haller, O., and Kochs, G. (2002). Interferon‐induced Mx proteins: Dynamin‐like GTPases with antiviral activity. Traffic 3, 710–717. Hopp, T. P. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204–1210. Janzen, C., Kochs, G., and Haller, O. (2000). A monomeric GTPase‐negative MxA mutant with antiviral activity. J. Virol. 74, 8202–8206. Kochs, G., Haener, M., Aebi, U., and Haller, O. (2002a). Self‐assembly of human MxA GTPase into highly‐ordered dynamin‐like oligomers. J. Biol. Chem. 277, 14172–14176. Kochs, G., and Haller, O. (1999a). GTP‐bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J. Biol. Chem. 274, 4370–4376. Kochs, G., and Haller, O. (1999b). Interferon‐induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc. Natl. Acad. Sci. USA 96, 2082–2086. Kochs, G., Janzen, C., Hohenberg, H., and Haller, O. (2002b). Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc. Natl. Acad. Sci. USA 99, 3153–3158.
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McNiven, M. A., Cao, H., Pitts, K. R., and Yoon, Y. (2000). The dynamin family of mechanoenzymes: Pinching in new places. Trends Biochem. Sci. 25, 115–120. Melen, K., Ronni, T., Broni, B., Krug, R. M., Vonbonsdorff, C. H., and Julkunen, I. (1992). Interferon‐induced Mx proteins form oligomers and contain a putative leucine zipper. J. Biol. Chem. 267, 25898–25907. Nakayama, M., Yazaki, K., Kusano, A., Nagata, K., Hanai, N., and Ishihama, A. (1993). Structure of mouse Mx1 protein: Molecular assembly and GTP‐dependent conformational change. J. Biol. Chem. 268, 15033–15038. Ponten, A., Sick, C., Weeber, M., Haller, O., and 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, 2591–2599. Praefcke, G. J., and McMahon, H. T. (2004). The dynamin superfamily: Universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell. Biol. 5, 133–147. Reichelt, M., Stertz, S., Krijnse‐Locker, J., Haller, O., and Kochs, G. (2004). Missorting of LaCrosse virus nucleocapsid protein by the interferon‐induced MxA GTPase involves smooth ER membranes. Traffic 5, 772–784. Richter, M. F., Schwemmle, M., Herrmann, C., Wittinghofer, A., and Staeheli, P. (1995). Interferon‐induced MxA protein: GTP binding and GTP hydrolysis properties. J. Biol. Chem. 270, 13512–13517. Schumacher, B., and Staeheli, P. (1998). Domains mediating intramolecular folding and oligomerization of MxA GTPase. J. Biol. Chem. 273, 28365–28370. Schwemmle, M., Richter, M. F., Herrmann, C., Nassar, N., and Staeheli, P. (1995). Unexpected structural requirements for GTPase activity of the interferon‐induced MxA protein. J. Biol. Chem. 270, 13518–13523. Steegmaier, M., Oorschot, V., Klumperman, J., and Scheller, R. H. (2000). Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics. Mol. Biol. Cell 11, 2719–2731. Taylor, G. A., Feng, C. G., and Sher, A. (2004). p47 GTPases: Regulators of immunity to intracellular pathogens. Nat. Rev. Immunol. 4, 100–109. Weber, F., Haller, O., and Kochs, G. (2000). MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J. Virol. 74, 560–563. Zhang, P., and Hinshaw, J. E. (2001). Three‐dimensional reconstruction of dynamin in the constricted state. Nat. Cell Biol. 3, 922–927. Zu¨ rcher, T., Pavlovic, J., and Staeheli, P. (1992). Mechanism of human MxA protein action: Variants with changed antiviral properties. EMBO J. 11, 1657–1661.
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[55] Assay and Functional Analysis of Dynamin‐Like Mx Proteins By GEORG KOCHS, MIKE REICHELT, DGANIT DANINO, JENNY E. HINSHAW , and OTTO HALLER Abstract
Mx proteins are interferon‐induced large guanosin triphosphatases (GTPases) that share structural and functional properties with dynamin and dynamin‐like proteins, such as self‐assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a 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 association of the human MxA GTPase with membranes of the endoplasmic reticulum seems to support its antiviral function by providing an interaction platform that facilitates viral target recognition, MxA oligomerization, and missorting of the resulting multiprotein complex into large intracellular aggregates. Introduction
Mx proteins are members of the superfamily of high molecular weight GTPases (Haller and Kochs, 2002). Many of these dynamin‐like GTPases localize to intracellular membranes and are involved in intracellular trafficking, membrane remodeling, and fission processes (Danino and Hinshaw, 2001; McNiven et al., 2000). Some family members appear to have a different role, in being key components of the early innate immune response against a wide variety of invading pathogens (Haller and Kochs, 2002; Praefcke and McMahon, 2004; Taylor et al., 2004). Accordingly, these GTPases are upregulated by type I ( /b) or type II ( ) interferons (IFN), and they function as intracellular resistance factors capable of restricting the growth of distinct pathogens. The Mx GTPases are expressed exclusively in IFN‐ /b–treated cells (Haller and Kochs, 2002). Human MxA, a 78 kDa protein, accumulates in the cytoplasm of IFN‐ treated cells and inhibits the replication of a wide range of viruses, including influenza viruses, measles virus, and bunyaviruses (Haller and Kochs, 2002). Biochemical and cell culture studies suggest that MxA interacts
METHODS IN ENZYMOLOGY, VOL. 404 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)04055-3
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directly with viral target structures. MxA was shown to bind to the nucleocapsids of Thogoto virus (THOV), an influenza‐like orthomyxovirus. As a consequence, the THOV nucleocapsids were prevented from entering the nucleus where transcription and replication of the viral genome normally occurs (Kochs and Haller, 1999b; Weber et al., 2000). MxA also inhibits the replication of La Crosse virus (LACV), a bunyavirus with a cytoplasmic replication phase. In this case, MxA binds to the viral nucleocapsid (N) protein and forms large copolymers that accumulate in the perinuclear area (Kochs et al., 2002b). Overall, MxA seems to act by interfering with the proper transport of critical viral components to their ultimate target compartments in infected cells. Membrane association and homo‐oligomerization are essential for the biological function of dynamin‐like proteins (Danino and Hinshaw, 2001; Praefcke and McMahon, 2004). MxA forms homo‐oligomers both in vitro and in vivo (Accola et al., 2002; Kochs et al., 2002a). Three distinct domains are involved in the self‐assembly process: (i) a ‘‘self‐assembly sequence’’ (SAS) that is located within the N‐terminal G‐domain and is conserved in all members of the dynamin‐like GTPases (Nakayama et al., 1993); (ii) a central interactive domain (CID) that mediates the association with the C‐terminal part of the molecule (Ponten et al., 1997); and (iii) a leucine zipper motif (LZ) at the extreme C‐terminus that interacts with the CID (Schumacher and Staeheli, 1998). The interaction between LZ and CID results in increased GTPase activity, indicating that the LZ region acts as a ‘‘GTPase effector domain’’ (GED) (Schwemmle et al., 1995), similar to the GED of dynamin. Furthermore, intra‐ and intermolecular interactions are critical for protein stability and recognition of viral target structures (Flohr et al., 1999; Janzen et al., 2000; Schwemmle et al., 1995). MxA protein localizes to a subcompartment of the smooth endoplasmic reticulum, suggesting that membrane binding and compartmentalization are important for its antiviral function (Accola et al., 2002; Reichelt et al., 2004). Our current model proposes that, in IFN‐treated cells, MxA forms large membrane‐associated self‐assemblies that serve as a stable storage pool from which MxA monomers are transiently released. The equilibrium between assembled and monomeric forms is presumably regulated by the GTPase activity. Upon infection, MxA monomers sense viral target structures and, by binding to them, form new assemblies involving specific viral components. Depending on the local concentration of the binding partners, more and more MxA molecules are recruited into these membrane‐associated copolymers, leading to mislocalization of the viral components and to viral inhibition (Haller and Kochs, 2002).
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Methods
Expression and Purification of MxA Recombinant human MxA is expressed in E. coli as an N‐terminal histidine‐tagged protein using the pQE‐9 vector system (Qiagen, Hilden, Germany), as described (Kochs et al., 2002a; Richter et al., 1995). E. coli strain M15 carrying the pQE‐9‐MxA expression plasmid is incubated at 28 in LB (Luria‐Bertani) medium containing 100 g/ml ampicillin and 25 g/ml kanamycin. At an optical density of 0.3 at 600 nm, MxA expression is induced by adding 0.03 mM isopropyl‐b‐D‐ thiogalactopyranoside followed by further incubation for 2 h. The cells are harvested at an optical density of 0.6 to 0.8 by centrifugation. The bacterial pellet can be stored at 70 . For lysis, the pellet from a 1 l culture is resuspended in 10 ml of buffer A (50 mM Tris, pH 8.0, 500 mM NaCl, 5 mM MgCl2, 0.1% NP40, 10% glycerol, 2 mM imidazole, and 7 mM b‐mercaptoethanol). Lysis is performed on ice by seven cycles of sonication for 30 s each with a Branson B15 sonifier. The homogenate is clarified by centrifugation for 25 min at 20,000g. The resulting supernatant (Fig. 1A and B, lanes 1) is incubated with 800 l Ni‐nitrilotriacetic acid‐ NTA agarose (Qiagen) by end‐over‐end rotation for 2 h at 4 . Then the agarose beads are poured into a column of 5 mm in diameter and washed with 30 ml of buffer A containing 30 mM imidazole followed by a 10 ml washing step with buffer B (like buffer A but with 100 mM NaCl and 2 mM imidazole). His‐MxA is eluted in 0.5 ml steps with buffer B containing 250 mM imidazole (Fig. 1A and B, lanes 2). For further purification and concentration, the Ni‐eluate is directly loaded on a Mono Q anion exchange column (1 ml, 5 50 mm; Amersham‐Pharmacia, Freiburg, Germany) equilibrated in buffer C (50 mM Tris, pH 8.0, 2 mM MgCl2, 100 mM NaCl, 10% glycerol, 0.05% NP40, 1.4 mM b‐mercaptoethanol). After washing with 5 ml of buffer C, His‐MxA is eluted with a linear gradient of 5 ml from 0.1 to 1 M NaCl in buffer C. His‐MxA typically elutes between 300 to 500 mM NaCl (Fig. 1A and B, lanes 3) with a protein concentration of about 1 mg/ml. The purified protein can be stored at 70 . The GTPase activity of MxA (the hydrolysis of GTP into GDP and inorganic phosphate) is measured as described (Richter et al., 1995). A GTPase inactive mutant with an amino acid exchange in the N‐terminal G‐domain, MxA(T103A) (Ponten et al., 1997), is used as a negative control. About 1 g of each purified protein is incubated in 50 l of buffer D (50 mM Tris, pH 8.0, 5 mM MgCl2, 100 mM KCl, 10% glycerol, 0.1 mM dithiothreitol (DTT)) with 1 mM GTP and [32P]‐ ‐GTP (200 nCi) in the
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FIG. 1. Purification and GTPase activity of MxA. (A) Coomassie blue‐stained SDS‐ polyacrylamide gel loaded with fractions obtained during the purification of His‐MxA expressed in E. coli. Lane 1, supernatant after centrifugation; lane 2, eluate from the Ni‐NTA affinity column; lane 3, eluate from the Mono Q ion exchange column. (B) Western blot analysis. One‐fifth of the fractions shown in (A) were blotted on a PVDF membrane and analyzed using the monoclonal anti‐MxA antibody M143 (dilution 1:500) (Flohr et al., 1999). (C) GTPase activity of recombinant wild‐type MxA and MxA(T103A), a GTPase‐ inactive mutant (Ponten et al., 1997). Thin‐layer chromatogram of GTP and GDP after incubation with Mono Q‐purified wild‐type MxA or MxA(T103A) (1 mg) for 0, 30, and 60 min at 37 .
presence of 100 nM AMP‐PNP. At various time points the reaction is terminated by mixing 10 l of the reaction mixture with the same volume of 2 mM ethylenediaminetetraacetic acid (EDTA) in 0.5% sodium dodecyl sulfate (SDS). To separate the radio‐labeled GDP product from the GTP substrate, 1.5 l of the stopped reaction is spotted onto a polyethyleneimine‐cellulose thin‐layer chromatography plate (PEI‐5725, Merck, Darmstadt, Germany) and developed in 1 M LiCl and 1 M acetic acid. Figure 1C
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shows an autoradiography of a dried plate. The result demonstrates that Mx proteins, in contrast to small Ras‐like GTPases, exhibit a high intrinsic GTPase activity. Oligomerization of MxA MxA Oligomerization in Living Cells Mx‐proteins form high molecular weight oligomers in vivo and in vitro as shown by chemical cross‐linking, gel filtration, and ultrastructural analysis of recombinant proteins (Kochs et al., 2002a; Melen et al., 1992; Richter et al., 1995). To monitor their self‐assembly in living cells, we established a nuclear translocation assay (Ponten et al., 1997). The idea of this assay is that in case of homooligomerization, an artificial nuclear form of MxA should be able to drag the normally cytoplasmic MxA protein into the nucleus. For this approach, MxA is modified by a foreign nuclear translocation signal (NLS). The NLS of the large T‐antigen of SV40 is fused to the N terminus of MxA resulting in TMxA that translocates into the nucleus when expressed in eukaryotic cells (Zu¨ rcher et al., 1992). To distinguish nuclear TMxA from the normal cytoplasmic MxA, wild‐type MxA is N‐terminally tagged with a FLAG peptide (Hopp, 1988). Mouse 3T3 cells are seeded onto glass coverslips. After 4 h, the cells are transfected with pHMG‐expression plasmids using 2 l of Lipofectamine transfection reagent (Invitrogen, Karlsruhe, Germany). At 20 h post‐transfection the cells are fixed with 3% paraformaldehyde (PFA) in phosphate‐buffered saline (pH 7.4) (PBS), permeabilized with 0.5% Triton X‐100, and stained using the monoclonal anti‐Mx antibody M143 (Flohr et al., 1999) or a monoclonal anti‐FLAG antibody (Sigma) in PBS with 5% fish gelatin. After three washing steps with PBS, the antigen‐bound primary antibodies are detected with fluorophore (Cy2)‐conjugated donkey antibodies (Dianova, Hamburg, Germany) and analyzed with a fluorescence microscope. Figure 2A shows that FLAG‐tagged wild‐type MxA accumulates in cytoplasmic dots (left panel) and TMxA in the nucleus (right panel). Upon coexpression of both cDNAs, the wild‐type MxA, detected by the FLAG antibody, predominantly accumulates in the nucleus, indicating a tight association with TMxA (Fig. 2A, middle panel). Interestingly, a fraction of FLAG‐MxA remains in the cytoplasm, presumably bound to intracellular membranes (see following). This approach might also be suitable to study protein‐protein interactions of other candidate proteins in living cells.
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FIG. 2. MxA oligomerization in vivo and in vitro. (A) TMxA and FLAG‐tagged wild‐type MxA were transfected either separately (left and right) or together (cotransfection, middle panel) in murine 3T3 cells. The subcellular localization of the recombinant proteins was analyzed by indirect immunofluorescence using either the anti‐FLAG or the anti‐MxA antibody M143 (dilution 1:500). (B) Cryo‐TEM images of MxA self‐assemblies. Purified His‐ MxA dialyzed overnight in low salt buffer with 1 mM GMP‐PCP self‐assembled into rings and open arcs (a). (b) Higher magnification of the rings revealed a structure of two parallel sets of electron dense globular domains (double arrowheads). (c) When dialyzed in the presence of GDP/BeF, MxA self‐organized into long, straight, and ordered complexes (arrows). (Bars ¼ 50 nm).
Structural Analysis of MxA Oligomers by Cryo‐transmission Electron Microscopy In vitro, MxA forms ring‐like and helical oligomers, similar to the structures formed by dynamin (Accola et al., 2002; Kochs et al., 2002a). These structures are studied at higher resolution using transmission electron microscopy at cryogenic temperatures (cryo‐TEM). Cryo‐TEM is based on ultra‐rapid thermal fixation of the sample that preserves the structures at their native state (Danino et al., 1997, 2004). Therefore, it is a powerful method, better suited to study Mx oligomer formation than conventional chemical fixation methods like negative staining.
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Specimens for cryo‐TEM are prepared at a controlled temperature of 25 and saturated water atmosphere in a controlled environment vitrification system. An 8 l drop of purified His‐MxA (1 g/l) in buffer E (20 mM Hepes, pH 7.2, 2 mM EGTA, 1 mM MgCl2, 1 mM DTT, 50 mM NaCl) and 1 mM nucleotide is placed on a TEM grid covered with perforated carbon film (Ted Pella, USA) held by a tweezers. The drop is blotted with a filter paper forming a film of 100–250 nm in thickness, and dropped into liquid ethane at its freezing point of 183 . The specimen is then transferred to liquid nitrogen for storage. Specimens are examined in a Tecnai G2 T12 transmission electron microscope operating at 120 kV using a Gatan 626 cryo holder maintained at below 175 . Imaging is done at low dose exposures to minimize radiation damage of the specimens, and images are recorded digitally on a Gatan 791 wide‐angle cooled CCD camera at effective magnifications of up to 50,000 using specific imaging procedures as described (Danino et al., 2001). Ring‐like MxA structures form upon dialysis of MxA in the presence of 1 mM GMP‐PCP, a nonhydrolyzing analogue of GTP (Fig. 2Ba). Higher magnification images reveal that the rings are composed of two parallel sets of electron dense globular domains that are formed most likely by intra‐ and interdomain associations of the MxA molecules (Fig. 2Bb). Likewise, long, ordered MxA assemblies displaying a ladder pattern are induced by dialysis of MxA against buffer E in the presence of GDP/BeF (1 mM GDP, 5 mM NaF, 500 mM BeCl2, in 5% ethylene glycol) for 20 h at 4 (Fig. 2Bc). Binding of GDP/BeF mimics a transition state during GTP hydrolysis (Ahmadian et al., 1997). This indicates that cryo‐TEM is possibly the method of choice to study the structure and the kinetics of assembly of high molecular weight Mx oligomers. Membrane Association of MxA Interestingly, human MxA is a membrane‐associated large GTPase, although it lacks obvious membrane interaction domains comparable to the PH domain of dynamin (Accola et al., 2002; Reichelt et al., 2004). To study the association of MxA with membranes, Vero cells constitutively expressing human MxA (Frese et al., 1995) are analyzed by differential centrifugation. For this, VA3 cells from two 150 cm2 petri dishes are lysed in 1 ml of buffer F (25 mM Hepes, pH 7.5, 2.5 mM MgCl2, 150 mM NaCl, 1 mM DTT) using a dounce‐homogenizer. The homogenate is centrifuged at 1000g (P1) and the resulting supernatant is subjected first to 10,000g (P10) and then to 100,000g centrifugation (P100 and S100). The pellets are resuspended in buffer F and equal amounts of protein are analyzed by Western blotting using the monoclonal anti‐Mx antibody M143
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FIG. 3. MxA is a membrane‐resident protein. (A) Fractionation of MxA‐expressing cells. Vero cells constitutively expressing high levels of recombinant human MxA (VA3) were compared to control cells lacking MxA (VN36) (Frese et al., 1995). Lysates of VA3 cells were centrifuged first at 1000g (P1), then at 10,000g (P10), and the resulting supernatant at 100,000g (P100 and S100). Equivalent amounts of protein were analyzed by Western blotting using the monoclonal anti‐MxA antibody M143. (B) In vitro interaction of purified His‐MxA with liposomes. MxA was incubated with PS‐liposomes for 1 h at 37 in the presence of GTP and then analyzed by cryo‐TEM. MxA assembles on the surface of a lipid vesicle (a). In the presence of lipid tubes MxA forms ring‐like assemblies (b) with a characteristic Y‐like appearance (arrows). Scale bar ¼ 100 nm.
(Fig. 3A). Approximately 30% of the total MxA protein content is sedimented with the microsomal pellet, P100, suggesting that part of MxA is associated with membranes.
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Structural Analysis of MxA‐membrane Association In Vitro To study in vitro association of MxA with membranes, a 10 g/l solution of phosphatidylserine (PS, Avanti Polar Lipids, Alabaster, AL) in chloroform is dried under a stream of nitrogen, kept under vacuum overnight, and resuspended to a final concentration of 2 g/l in buffer E. Liposomes are generated by extruding the lipid solution 15 times through a 1 m polycarbonate membrane (Avanti Polar Lipids). Purified MxA is incubated with the liposomes (final concentrations 0.3 and 0.25 g/l for MxA and PS, respectively) for 1 h at 37 in the presence of 1 mM GTP, and is subsequently prepared for cryo‐TEM as described above. Figure 3Ba shows that MxA associates with the surface of liposomes, indicating that MxA has the potential to directly interact with lipid membranes. In the presence of lipid tubes, sometimes the formation of ring‐like MxA structures can be detected. At high magnification, these assemblies show an Y‐like shape (Fig. 3Bb), reminiscent of the Y‐shaped assemblies of dynamin around lipid tubes (Zhang and Hinshaw, 2001). MxA Interaction with Viral Target Structures According to our hypothesis, the antiviral action of Mx proteins is based on their direct interaction with viral components essential for gene expression and genome replication (Haller and Kochs, 2002; Kochs and Haller, 1999a; Kochs et al., 2002b). To show direct interaction of human MxA with viral structures, we analyze infected cells by immunofluorescence and coimmunoprecipitation. VA3 cells are seeded on glass coverslips and infected with 10 plaque‐ forming units per cell of La Crosse virus (LACV), a MxA‐sensitive bunyavirus (Frese et al., 1996). After 16 h the cells are stained for triple fluorescence analysis as described above, using the monoclonal anti‐MxA antibody M143, a goat polyclonal anti‐Syntaxin17 antiserum, and a polyclonal rabbit antiserum directed against the nucleoprotein (N) of LACV. Syntaxin17 is a marker for a subcompartment of the smooth endoplasmic reticulum (ER) (Steegmaier et al., 2000). The primary antibodies are detected with fluorophore (Cy2, Cy3, and Cy5)‐conjugated donkey secondary antibodies, respectively, and analyzed with a Leica TCSSP2 confocal laser scanning microscope. Colocalization of MxA with Syntaxin17 clearly shows that MxA is a membrane‐associated protein with a distinct subcellular distribution (Fig. 4Aa and b). The colocalization of MxA with the viral N protein indicates a tight association of membrane‐resident MxA with viral nucleocapsids (Fig. 4Aa and c).
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FIG. 4. MxA forms complexes with viral target structures and cellular membranes. (A) Colocalization of MxA/N protein complexes with smooth ER membranes. VA3 cells were infected with LACV for 16 h, fixed as described above, and stained with monoclonal anti‐ MxA antibody (a, green), goat anti‐Syntaxin17 antibody (b, red), and a rabbit antiserum specific for the viral N protein (c, blue). The primary antibodies were detected with fluorophore (Cy2, Cy3, and Cy5)‐conjugated secondary antibodies, respectively. The pictures were recorded using a Leica TCSSP2 confocal laser scanning microscope. Bar ¼ 8 mm. (B) VA3 cells were infected with LACV or left uninfected. Cells were lysed in the presence or absence of GTP‐gS and subjected to immunoprecipitation using the N‐specific antiserum. Bound MxA was detected by Western blotting using the monoclonal anti‐MxA antibody M143 (from Kochs et al., 2002, # 2002).
Coimmunoprecipitation of MxA with Viral N Protein To verify the MxA/N interaction, virus‐infected VA3 cells from one 150 cm2 petri dish are lysed as described above in buffer G (50 mM Tris, pH 7.5, 0.1% Nonidet P‐40, 5 mM MgCl2, 0.5 mM DTT) in the presence or absence of 200 mM guanosine 50 ‐O‐[ ‐thio]‐triphosphate (GTP‐ S), a nonhydrolyzable GTP analogue. The lysates are incubated with 2 l of the polyclonal rabbit anti‐N antiserum coupled to 30 l of protein A‐sepharose beads (Amersham‐Pharmacia) for 2 h at 4 . After washing the beads
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several times in buffer G, bound proteins are eluted by incubation in SDS‐ containing Laemmli sample buffer for 5 min at 95 and analyzed by Western blotting using the monoclonal anti‐MxA antibody M143. The Western blot (Fig. 4B) demonstrates that MxA interacts with the viral N protein only in the presence of GTP‐ S, indicating that MxA is competent for interaction in its GTP‐bound conformation (Kochs et al., 2002b).
References Accola, M. A., Huang, B., Masri, A. A., and McNiven, M. A. (2002). The antiviral dynamin family member, MxA, tubulates lipids and localizes to the smooth endoplasmic reticulum. J. Biol. Chem. 277, 21829–21835. Ahmadian, M. R., Mittal, R., Hall, A., and Wittinghofer, A. (1997). Aluminium fluoride associates with the small guanine nucleotide binding proteins. FEBS Lett. 408, 315–318. Danino, D., Bernheim‐Groswasser, A., and Talmon, Y. (2001). Digital cryogenic transmission electron microscopy: An advanced tool for direct imaging of complex fluids. Colloids and Surfaces A: Physicochem. Engineer. Aspects 183, 113–122. Danino, D., and Hinshaw, J. E. (2001). Dynamin family of mechanoenzymes. Curr. Opinion Cell Biol. 13, 454–460. Danino, D., Kaplun, A., Lindblom, G., Rilfors, L., Oradd, G., Hauksson, J. B., and Talmon, Y. (1997). Cryo‐TEM and NMR studies of a micelle‐forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B. Chem. Phys. Lipids 85, 75–89. Danino, D., Moon, K. H., and Hinshaw, J. E. (2004). Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin. J. Struct. Biol. 147, 259–267. Flohr, F., Schneider‐Schaulies, S., Haller, O., and 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, 24–28. Frese, M., Kochs, G., Feldmann, H., Hertkorn, C., and Haller, O. (1996). Inhibition of Bunyaviruses, Phleboviruses, and Hantaviruses by human MxA protein. J. Virol. 70, 915–923. Frese, M., Kochs, G., Meier‐Dieter, U., Siebler, J., and Haller, O. (1995). Human MxA protein inhibits tick‐borne Thogoto virus but not Dhori virus. J. Virol. 69, 3904–3909. Haller, O., and Kochs, G. (2002). Interferon‐induced Mx proteins: Dynamin‐like GTPases with antiviral activity. Traffic 3, 710–717. Hopp, T. P. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204–1210. Janzen, C., Kochs, G., and Haller, O. (2000). A monomeric GTPase‐negative MxA mutant with antiviral activity. J. Virol. 74, 8202–8206. Kochs, G., Haener, M., Aebi, U., and Haller, O. (2002a). Self‐assembly of human MxA GTPase into highly‐ordered dynamin‐like oligomers. J. Biol. Chem. 277, 14172–14176. Kochs, G., and Haller, O. (1999a). GTP‐bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J. Biol. Chem. 274, 4370–4376. Kochs, G., and Haller, O. (1999b). Interferon‐induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc. Natl. Acad. Sci. USA 96, 2082–2086. Kochs, G., Janzen, C., Hohenberg, H., and Haller, O. (2002b). Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc. Natl. Acad. Sci. USA 99, 3153–3158.
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McNiven, M. A., Cao, H., Pitts, K. R., and Yoon, Y. (2000). The dynamin family of mechanoenzymes: Pinching in new places. Trends Biochem. Sci. 25, 115–120. Melen, K., Ronni, T., Broni, B., Krug, R. M., Vonbonsdorff, C. H., and Julkunen, I. (1992). Interferon‐induced Mx proteins form oligomers and contain a putative leucine zipper. J. Biol. Chem. 267, 25898–25907. Nakayama, M., Yazaki, K., Kusano, A., Nagata, K., Hanai, N., and Ishihama, A. (1993). Structure of mouse Mx1 protein: Molecular assembly and GTP‐dependent conformational change. J. Biol. Chem. 268, 15033–15038. Ponten, A., Sick, C., Weeber, M., Haller, O., and 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, 2591–2599. Praefcke, G. J., and McMahon, H. T. (2004). The dynamin superfamily: Universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell. Biol. 5, 133–147. Reichelt, M., Stertz, S., Krijnse‐Locker, J., Haller, O., and Kochs, G. (2004). Missorting of LaCrosse virus nucleocapsid protein by the interferon‐induced MxA GTPase involves smooth ER membranes. Traffic 5, 772–784. Richter, M. F., Schwemmle, M., Herrmann, C., Wittinghofer, A., and Staeheli, P. (1995). Interferon‐induced MxA protein: GTP binding and GTP hydrolysis properties. J. Biol. Chem. 270, 13512–13517. Schumacher, B., and Staeheli, P. (1998). Domains mediating intramolecular folding and oligomerization of MxA GTPase. J. Biol. Chem. 273, 28365–28370. Schwemmle, M., Richter, M. F., Herrmann, C., Nassar, N., and Staeheli, P. (1995). Unexpected structural requirements for GTPase activity of the interferon‐induced MxA protein. J. Biol. Chem. 270, 13518–13523. Steegmaier, M., Oorschot, V., Klumperman, J., and Scheller, R. H. (2000). Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics. Mol. Biol. Cell 11, 2719–2731. Taylor, G. A., Feng, C. G., and Sher, A. (2004). p47 GTPases: Regulators of immunity to intracellular pathogens. Nat. Rev. Immunol. 4, 100–109. Weber, F., Haller, O., and Kochs, G. (2000). MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J. Virol. 74, 560–563. Zhang, P., and Hinshaw, J. E. (2001). Three‐dimensional reconstruction of dynamin in the constricted state. Nat. Cell Biol. 3, 922–927. Zu¨ rcher, T., Pavlovic, J., and Staeheli, P. (1992). Mechanism of human MxA protein action: Variants with changed antiviral properties. EMBO J. 11, 1657–1661.