Intramolecular Complementing Mutations in Tobacco Mosaic Virus Movement Protein Confirm a Role for Microtubule Association in Viral RNA

Transport
Vitaly Boyko, Jamie Alan Ashby, Elena Suslova, Jacqueline Ferralli, Oliver Sterthaus, Carl M. Deom and Manfred Heinlein J. Virol. 2002, 76(8):3974. DOI: 10.1128/JVI.76.8.3974-3980.2002. Downloaded from http://jvi.asm.org/ on November 25, 2013 by guest

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JOURNAL OF VIROLOGY, Apr. 2002, p. 39743980 0022-538X/02/$04.000 DOI: 10.1128/JVI.76.8.39743980.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 8

Intramolecular Complementing Mutations in Tobacco Mosaic Virus Movement Protein Conrm a Role for Microtubule Association in Viral RNA Transport
Vitaly Boyko,1 Jamie Alan Ashby,1 Elena Suslova,1 Jacqueline Ferralli,1 Oliver Sterthaus,1 Carl M. Deom,2 and Manfred Heinlein1*
Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, CH-4058 Basel, Switzerland,1 and Department of Plant Pathology, The University of Georgia, Athens, Georgia 306022
Received 2 November 2001/Accepted 22 January 2002

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The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green uorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MPP81S-GFP and MPP81S;T104I;R167K-GFP differ in subcellular distribution. MPP81S-GFP lacks specic sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MPP81S;T104I;R167K-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations conrm a role for microtubules in TMV RNA cell-to-cell transport. Plant virus movement is an active process that depends on viral-encoded movement proteins (MP) to facilitate intercellular transport of the viral genomes through plasmodesmata (Pd), channels in the cell wall that provide symplastic connections between adjacent cells. Numerous MPs have been reported to increase the permeability of Pd and to be able by themselves to move between cells. However, an increasing number of reports indicate that gating of Pd is insufcient to allow infection to spread into adjacent cells (8, 10, 49) and thus imply that infection depends on additional MP-mediated mechanisms. One of the most studied MPs is that of Tobacco mosaic virus (TMV) (4, 15, 21). The MP of TMV accumulates in Pd (2, 22, 45, 60) and increases their size exclusion limit (48, 66). In addition, MP binds both RNA and single-stranded DNA in vitro (16, 17), suggesting that the MP may chaperone viral RNA in vivo. The formation of viral ribonucleoprotein complexes (vRNPs) is supported by the ability of microinjected MPs to mediate the transport of coinjected nucleic acids (23, 28, 46, 62). The existence of vRNPs is also supported by biochemical studies (25, 26, 34) as well as by elegant microinjection experiments indicating that MP functions in vivo as a cis-acting mediator of plasmodesmal transport, requiring its physical association with the transported molecule (63). To further elucidate the cellular mechanism by which MP facilitates the spread of viral RNA (vRNA) or the proposed vRNP from virus-replicating cells into adjacent cells, several research groups have undertaken the characterization of host components that interact with TMV MP and have shown that
* Corresponding author. Mailing address: Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Phone: 41 61 697 85 17. Fax: 41 61 697 39 76. E-mail: heinlein@fmi.ch. 3974

a fusion between MP and green uorescent protein (GFP) accumulates in Pd (31, 48) and also colocalizes with components of the cytoskeleton (30, 44) and endoplasmic reticulum (ER)-derived membranes (31, 54). During infection of leaf epidermal cells or protoplasts derived from tobacco suspension cell line BY-2, ER membranes form aggregates or inclusion bodies (31), and MP seems to be involved in this process (54). Studies using BY-2 protoplasts have shown that the bodies contain replicase (31) and vRNA (42) in addition to MP, thus providing evidence that these bodies represent sites of virus replication and protein synthesis. The presence of MP in inclusion bodies is consistent with MP synthesis at these sites. MP-GFP exhibits strong accumulation in inclusion bodies under conditions when intercellular spread of vRNA is slow (9), suggesting that the effective turnover or removal of MP from replication sites acts as an important determinant for efcient vRNA transport. In fact, during the course of infection, MPGFP produced in these sites is conveyed onto microtubules (1, 31). MP also appears to interact with actin (44), and since actin and myosin are components of Pd (6, 53, 56, 65), it is conceivable that microlaments and microtubules share synergistic functions in the spread of vRNA (14, 68). However, whereas an active role for actin laments in vRNA movement has yet to be demonstrated, several recent studies support the functional signicance of microtubule-associated MP. Infected BY-2 protoplasts were used to demonstrate that vRNA localizes to microtubules in an MP-dependent manner (42) and shows aberrant localization with a mutant MP that fails to associate with microtubules (43). In planta observations indicated a positive correlation between the efciency by which infection spreads from cell to cell and the visualization of MP-GFP-associated microtubules in cells at the leading front

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of infection (9). A direct correlation between the ability of MP to associate with microtubules and to facilitate the transport of vRNA was also demonstrated by the in planta analysis of TMV mutants carrying either internal (TAD5) (33, 43) or C-terminal (10) deletion mutations in MP. A role for microtubules in vRNA transport is also supported by observations indicating that a functionally defective MP which confers virus resistance to plants if expressed from a transgene (MP-derived resistance) (39) interferes with microtubule association of wild-type MP (37). Further evidence implicating a role for microtubules in vRNA movement was provided recently by the identication of a conserved sequence motif in tobamovirus MP that shares similarity with a region in tubulins that is thought to mediate lateral contacts between microtubule protolaments (8). The signicance of this nding was corroborated by point mutations in this motif that conferred temperature sensitivity to microtubule association and to vRNA intercellular transport functions of MP (8). These observations also suggested that MP may mimic tubulin-tubulin interfaces for direct interactions with the microtubule lattice (8). Consistently, it was found that MP associates with microtubules independent of virus infection (31, 37, 54) and of any plant-specic factor (8). Moreover, the protein coprecipitates with microtubules (8), and MP-associated microtubule complexes are highly stable against salts or other microtubule-disrupting conditions (8). Here, we provide further supporting evidence for the involvement of MP-microtubule complexes in vRNA transport. It was previously reported that a dysfunctional mutation in MP is reverted by second-site mutations in the protein (19). Our ndings demonstrate that this intramolecular complementation event is correlated with the restoration of the microtubule binding capacity of MP. Moreover, the results suggest that microtubule association as well as the function of MP might depend on a fold in which the three complementing amino acid positions are in close proximity.
MATERIALS AND METHODS Constructs. Plasmid pTf5-nx1 was used as a template for site-directed mutagenesis as described in detail elsewhere (8, 10). Infectious viral RNA of TMVMP:GFP5, TMV-MPP81S-GFP, and TMV-MPP81S;T104I;R167K-GFP was produced by in vitro transcription of plasmids pTf5-nx2 (10), pTf5-PS, and pTf5PSTIRK, respectively, using a MEGAscript T7 kit (Ambion). Inoculation of plants and protoplasts. Nicotiana tabacum cv. Xanthi NN and Nicotiana benthamiana plants (5 to 6 weeks old) were mechanically inoculated (in the presence of Carborundum) with transcripts derived from in vitro reactions, and the plants were maintained in 70% humidity at 22C during the 16-h photoperiod and at 20C during the dark period. Protoplasts of tobacco suspension cell line BY-2 were prepared and inoculated by electroporation with infectious transcripts as described elsewhere (64). Following inoculation, protoplasts were resuspended in 10 ml of medium and cultured as 2-ml aliquots in 35-mmdiameter petri dishes in the dark at 28C. Actinomycin D (30 g/ml) was added to the protoplasts to increase MP expression (7, 31). Microscopy. Protoplasts were harvested at 20 h postinfection (hpi), xed for 30 min in phosphate-buffered saline (pH 7.4) containing 3% paraformaldehyde and 5 mM EGTA, and then spun on polylysine-coated slides and dried. The samples were then mounted in Mowiol (Calbiochem) containing 2.5% 1,4-diazobicyclo[2.2.2]-octane (DABCO) as an antifade reagent. Fluorescence microscopy was performed with a Nikon Eclipse E800 microscope equipped with CFI Plan Apochromat objectives (Nikon Corp., Tokyo, Japan) and an XF100 (Omega Optical, Inc., Brattleboro, Vt.) lter set for visualization of GFP uorescence. Protoplast uorescence was analyzed with 60 oil immersion lenses. Infection sites on leaves were viewed with 2 or 4 lenses. High-magnication microscopy of infection sites was performed by using leaf disks placed on glass slides and 100 oil immersion objectives. Images were acquired and processed using an

ORCA-100 progressive scan interline charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab 3 software (Improvision, Coventry, England). Production of wild-type and mutant MP for in vitro assays. Plasmids pTf5-nx2, pTf5-PS, and pTf5-PSTIRK were used as templates for amplication of each corresponding MP gene by PCR, using specic primers for the introduction of unique 5 Eco47III and 3 BamHI restriction sites for subcloning into expression vector pQE60 (Qiagen) that was predigested with NcoI, treated with Klenow enzyme for lling the resulting 5 overhang, and subsequently digested with BamHI. Ligation of pQE60 with the PCR-amplied Eco47III-BamHI restriction fragment resulted in expression constructs (designated pQ-MP:H6, pQ-PS:H6 and pQ-PSTIRK:H6, respectively) with an ATG start codon corresponding to the rst methionine of MP and the 3 terminus of MP fused to six histidine codons immediately upstream of a TAA stop codon. The integrity of each construct was determined by DNA sequence analysis and recombinant plasmids were used to transform Escherichia coli strain M15[pRep4] (Qiagen). Ten milliliters of LuriaBertani medium supplemented with 50 g of carbenicillin per ml and 25 g of kanamycin per ml was inoculated with single colonies and bacterial cultures were grown overnight at 37C with agitation (230 rpm). Expression cultures were initiated by inoculating 400 ml of the above medium with 5 ml of overnight culture and cells were grown until the optical density at 600 nm reached 0.8 (3 h). Expression of MP:His6wt, MP:His6P81S, and MP:His6P81S;T104I;R167K was induced by the addition of 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) into each of the cultures, which were subsequently grown for an additional 3 h before cells were harvested by centrifugation at 10,000 g for 10 min at 4C and cell pellets were frozen in liquid nitrogen. MP:His6wt, MP:His6P81S, and MP:His6P81S;T104I;R167K were prepared by a procedure adapted from that of Brill et al. (13) which yielded milligram quantities of the proteins from insoluble inclusion bodies. Fifteen milliliters of ice-cold lysis buffer (20 mM Tris-Cl [pH 8.0], 30 U of DNase I, 10 g of RNase A per ml, and protease inhibitor cocktail [Roche]) was added to the frozen bacterial pellets, which were subsequently thawed on ice-water. Cells were lysed by repeated sonication (50 W) on ice (Labsonic 2000; B. Braun, Melsungen, Germany) followed by another cycle of freezing in liquid nitrogen and thawing on ice-water. After 10 min, the insoluble inclusion bodies were pelleted by centrifugation at 13,000 g for 15 min in a Sorvall SA 600 rotor at 4C. Inclusion body pellets were resuspended in 10 ml of ice-cold wash buffer (20 mM Tris-Cl [pH 8.0], 2 M urea, 0.5 M NaCl, 2% [vol/vol] Triton X-100, and protease inhibitor cocktail) by sonication (on ice) and incubation on ice for 5 min, followed by centrifugation as described above. Washing and pelleting of inclusion bodies were repeated until contaminating material could no longer be observed in the sediments (typically three times). The inclusion bodies were then denatured by sonication in 5 ml of solubilization buffer (SB buffer) (20 mM Tris-Cl [pH 8.0], 8 M urea, 0.5 M NaCl, and 5 mM imidazole) and overnight shaking at 25C, followed by centrifugation at 20,000 g for 15 min at 4C. The proteins were then puried by Ni2-nitrilotriacetic acid (NTA) afnity chromatography using 1-cm-diameter columns that were packed with 4 ml of activated NTA-Ni2 slurry (Qiagen) and equilibrated with 10 column volumes of SB buffer. Claried protein solutions were allowed to enter the gel bed by gravity ow and columns were washed with 15 volumes of SB buffer containing 20 mM imidazole. The proteins were eluted into SB buffer containing 500 mM imidazole and eluent fractions were analyzed for purity and yield by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (38) and Coomassie blue staining. Aliquots (1 ml) of puried proteins were dialyzed against 100 ml of double-distilled water (ddH2O) at room temperature for 1 h. Proteins were subsequently dialyzed twice against 4 liters of ddH2O at 4C with 6-h intervals. Protein solutions were cleared of insoluble material by centrifugation at 20,000 g for 30 min (4C) in a desktop microcentrifuge, followed by centrifugation at 100,000 g for 1 h in a Beckman TLA-100 ultracentrifuge at 4C. A protein concentration assay was performed (11) and solutions were diluted to 0.1 to 0.3 mg/ml with ddH20. One molar Tris-Cl buffer, pH 7.5, was added to each protein sample to a nal concentration of 10 mM, and samples were equilibrated on ice for 2 h. Samples were again cleared by ultracentrifugation and assayed for protein concentration before aliquots were rapidly frozen in liquid nitrogen and stored at 80C until use. In vitro RNA-binding assay. [-32P]UTP-labeled RNA was produced by in vitro transcription (MAXIscript T7 kit; Ambion Inc.) using a PCR-derived DNA template corresponding to the 5-terminal region of the GFP gene and containing a synthetic 5-proximal T7 promoter sequence. Transcripts (nucleotides 1 to 123) were puried by 6% denaturing polyacrylamide gel electrophoresis (59) and resuspended in ddH2O (specic activity, 1.1 107 cpm/g of RNA). The in vitro RNA-binding assay was performed by incubation of 0.1 pmol (4.4 ng) of RNA probe with increasing amounts of each of the proteins (MP:H6, PS:H6, and PSTIRK:H6 in 10 mM Tris-Cl [pH 7.5]) for 30 min at 4C in a reaction mixture

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FIG. 1. Local lesion assay on leaves of N. tabacum cv. Xanthi NN to test for infectivity and cell-to-cell movement functions of TMV-MPP81SGFP and TMV-MPP81S;T104I;R167K-GFP. (a) TMV-MPP81S-GFP does not give rise to local lesions. (b) TMV-MPP81S-GFP produces local lesions on plants that are transgenic for MP. (c) TMV-MPP81S;T104I;R167K-GFP triggers the formation of small local lesions on wild-type plants.

(10 l) containing 10 mM Tris-Cl, pH 7.5, and 0.1% (vol/vol) Triton X-100. Samples were then loaded onto 1% nondenaturing agarose gels (in 45 mM Tris-borate [pH 8.0] and 0.1% [vol/vol] Triton X-100) and electrophoresed for 2 h at 6.5 V/cm. Gels were dried at 80C and exposed to X-ray lm for 3 h at 75C.

RESULTS Functional complementation of P81S by T104I and R167K. It was reported previously that a defect in vRNA movement caused by the P81S mutation in MP is restored by additional T104I and R167K mutations in the protein (19). To correlate this intramolecular complementation event with subcellular localization of MP, the mutations were engineered into the MP of TMV-MP-GFP5 (10). The resulting virus constructs are referred to as TMV-MPP81S-GFP and TMV-MPP81S;T104I;R167KGFP, respectively. To test if the three mutations complement to cause function in the context of the MP-GFP fusion protein, we inoculated leaves of N. tabacum cv. Xanthi NN with infectious transcripts of the viral constructs. N. tabacum cv. Xanthi NN plants harbor the N gene which triggers a hypersensitive cell death response in infected cells, resulting in the formation of local necrotic lesions. As shown in Fig. 1, no local lesion formation was observed when leaves were inoculated with TMV-MPP81S-GFP (Fig. 1a). However, the same virus was infectious on N. tabacum cv. Xanthi NN plants carrying a transgene for MP (line 2005 [20]) (Fig. 1b), indicating that this virus is infectious but encodes a defective MP and therefore requires complementation by wild-type MP for cell-tocell movement. In contrast to TMV-MPP81S-GFP, TMVMPP81S;T104I;R167K-GFP gave rise to lesion formation on wildtype N. tabacum cv. Xanthi NN plants (Fig. 1c), demonstrating that this virus encodes a functional MP-GFP. The small lesion size is consistent with the small size and delayed occurrence of lesions produced by the original revertant (19) and indicates that the triple mutant functions less efciently than wild-type MP-GFP. Collectively, these tests demonstrated that MPP81SGFP and MPP81S;T104I;R167K-GFP reect the activity proles reported for the unfused MP mutants (19). Subcellular localization of MPP81S-GFP and MPP81S;T104I;R167K-

GFP in infected protoplasts. To correlate the activity proles of mutant MP-GFP proteins with their specic subcellular sites of accumulation, we rst analyzed infected BY-2 protoplasts. TMV-MPP81S-GFP gave rise to infection in protoplasts, conrming that this virus can replicate and that the lack of infection in N. tabacum cv. Xanthi NN plants is caused by dysfunctional movement. Microscopic analysis of infected protoplasts at 20 hpi revealed no patterns of specic accumulation of MPP81S-GFP (Fig. 2A), indicating that this mutant MP fails to associate with subcellular components. In contrast, protoplasts infected with TMV-MPP81S;T104I;R167K-GFP (Fig. 2B) produced uorescence patterns indicating that MPP81S;T104I;R167K-GFP accumulates in inclusion bodies (panel a), on microtubules (panel b), in peripheral puncta (panel c), and as hair-like protrusions on the surface of the protoplasts (panel d). These patterns of accumulation are very similar, if not identical, to the range of patterns reported for protoplasts infected with wild-type TMV-MP-GFP (31). Subcellular localization of MPP81S-GFP and MPP81S;T104I;R167KGFP during infection in N. benthamiana leaves. The subcellular distribution of MPP81S-GFP and MPP81S;T104I;R167K-GFP in epidermal cells was analyzed in infection sites formed on leaves of N. benthamiana, a systemic host of TMV (Fig. 3). As expected from our functional assay in N. tabacum cv. Xanthi NN plants (Fig. 1), TMV-MPP81S-GFP produced uorescent infection sites (Fig. 3a) only in plants that were transgenic for wild-type MP. High-magnication microscopy revealed the localization of MPP81S-GFP at punctate sites near or within the cell wall, presumably in Pd (Fig. 3b and c). No other subcellular site of accumulation could be detected. In contrast to TMV-MPP81S-GFP, TMV-MPP81S;T104I;R167K-GFP was infectious in wild-type as well as MP-transgenic plants and gave rise to ring-shaped infection sites (Fig. 3d) that were similar to those reported for wild-type TMV-MP-GFP (31), except that the size was smaller (data not shown). Microscopy revealed the localization of MPP81S;T104I;R167K-GFP in inclusion bodies (Fig. 3e), on microtubules (Fig. 3f), and in Pd (arrows in panels e and f). The pattern of subcellular localization of

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form migratory and nonmigratory complexes with RNA. Wildtype MP:His6wt (Fig. 4a) produces a stable and dened complex with RNA that maintains its migratory behavior in the gel-shift assay in the presence of increasing amounts of protein. The MP:His6P81S (Fig. 4b) and MP:His6P81S;T104I;R167K (Fig. 4c) mutant MPs also form migratory complexes. However, the RNA retardation patterns are more diffuse than with MP:His6wt (Fig. 4a), indicating that the mutant proteins form less-dened complexes with the offered RNA molecule. In addition, all three proteins form high-molecular-weight complexes with RNA that are unable to enter the gel. Consistent with previous reports (16, 34, 41), higher concentrations of MP (120 nM) resulted exclusively in the formation of nonmigratory complexes (not shown), which further supports the idea that MP can decorate RNA to high densities (36) by a cooperative process. Nonetheless, because the MP:His6P81S mutant protein is able to bind RNA per se and the apparent RNA mobility pattern produced by MP:His6P81S is very similar to that of the functional reversion mutant (MP:His6P81S;T104I;R167K), we can infer that the lack of MPP81S-GFP accumulation in inclusion bodies and on microtubules is not caused by a deciency in RNA-binding activity. DISCUSSION In the past few years, signicant progress has been made in understanding the cellular mechanism of TMV intracellular and intercellular movement (1, 4, 29, 40, 55). Particularly, the construction of infectious TMV derivatives that express MP as an MP-GFP fusion protein (27, 30, 44) paved the way for the identication and characterization of subcellular structures that contribute to the successful targeting and trafcking of the viral genome to Pd and into adjacent cells (31, 50). Microtubules emerged as key players (1, 8, 29), which may be consistent with their well-documented involvement in the trafcking of mRNAs, vesicles, viruses, and proteins in yeast and animal cells (3, 12, 32, 47, 51, 52, 57, 58). Considering the evidence for the trafcking of endogenous RNA species in plants (18, 35), microtubules may have roles beyond virus movement. In fact, the evolvement of MP might represent a viral adaptation to endogenous macromolecular trafcking mechanisms (62, 67). Studies on viral MP thus may reveal factors and cellular components of general importance in cell-to-cell and long distance signaling. To further investigate a role for microtubules in the trafcking of vRNA, we used a second-site revertant of a dysfunctional MP. Our results demonstrate a correlation between functional complementation and complementation with respect to subcellular localization. The dysfunctional MPP81SGFP has maintained the capacity to localize to Pd, adding to the already existing evidence that Pd localization and Pd gating alone is insufcient to allow the spread of vRNA (8, 10, 49). The functionally revertant MPP81S;T104I;R167K-GFP carrying the complementing mutations accumulates to inclusion bodies and microtubules in addition to Pd, suggesting that either one or both of these structures play essential roles during vRNA transport. Accumulation of MP-GFP to detectable levels in inclusion bodies, however, appears not to be required for vRNA movement, as was suggested by recent studies (9, 10). Accumulation

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FIG. 2. Patterns of subcellular distribution of MPP81S-GFP and MPP81S;T104I;R167K-GFP observed in infected protoplasts at 20 hpi. (A) TMV-MPP81S-GFP-infected protoplast. MPP81S-GFP does not localize to any discernible intracellular structure. (B) TMVMPP81S;T104I;R167K-GFP-infected protoplasts. MPP81S;T104I;R167K-GFP localizes to inclusion bodies (a), microtubules (b), peripheral puncta (c), and hair-like protrusions (d). Bar, 10 m.

MPP81S;T104I;R167K-GFP thus was the same as that described for wild-type MP-GFP (31). RNA-binding properties of MP:His6wt, MP:His6P81S, and MP:His6P81S;T104I;R167K. Since it has been proposed that MP mediates the transport of vRNA in the form of a vRNP, it is plausible that the association of MP with inclusion bodies and microtubules may depend on vRNA binding. The association defect of MPP81S-GFP could therefore be a consequence of abolished RNA-binding capacity. In order to test this possibility, we incubated puried MP (MP:His6wt, MP:His6P81S, and MP:His6P81S;T104I;R167K, respectively) with a 32P-labeled RNA (10 nM) and analyzed the resulting complexes by electrophoresis. As shown in Fig. 4, both the wild-type and mutant MPs

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FIG. 3. Subcellular distribution of MPP81S-GFP and MPP81S;T104I;R167K-GFP in leaf epidermal cells of N. benthamiana. (a) Infection site of TMV-MPP81S-GFP on MP-transgenic plant. (b and c) MPP81S-GFP accumulates only in Pd. (d) Infection site of TMV-MPP81S;T104I;R167K-GFP. (e) MPP81S;T104I;R167K-GFP in association with inclusion bodies and Pd (arrow). (f) MPP81S;T104I;R167K-GFP associated with microtubules and Pd (arrows). Bars, 1 mm (a and d) and 10 m (b, c, e, and f).

of MP-GFP in these sites was reduced under conditions that enhanced efcient vRNA movement (9). Moreover, accumulation of MP-GFP to visible levels in inclusion bodies was even abolished by deletion of amino acids 214 to 268 that are dispensable for MP function (5, 10). It thus appears unlikely that the reversion of the dysfunctional P81S mutation by intramolecular complementation is due to the restoration of the ability to accumulate in inclusion bodies. Rather, our observations further conrm existing evidence that the function of MP in viral movement depends on its ability to associate with microtubules (810). The functionally complementing mutations in MP may cause the restoration of microtubule association by

stabilizing the proper fold of the protein or by other indirect mechanisms. We considered the possibility that the P81S mutation might hinder binding of MP to vRNA that in turn could be a prerequisite for microtubule association. In fact, we reasoned that the restoration of accumulation of MP-GFP in inclusion bodies and on microtubules observed with the triple mutant might be caused by the restoration of RNA binding within ER-associated replication sites. However, the results of our in vitro RNA-binding assays argue against this possibility. We found that MP carrying the dysfunctional P81S mutation still binds RNA, as does the functionally restored triple mutant protein. Despite the fact that our in vitro RNA-binding con-

FIG. 4. RNA-binding properties of wild-type and mutant MPs puried from E. coli. 32P-labeled RNA (10 nM; see Materials and Methods) was incubated with increasing amounts of MP:His6wt (a), MP:His6P81S (b), and MP:His6P81S;T104I;R167K (c) as indicated. Resulting protein-RNA complexes were visualized by nondenaturing electrophoresis and autoradiography.

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ditions may not fully reect the in vivo situation, it appears unlikely that the aberrant distribution of MPP81S-GFP is caused by the inability to form a vRNP. The mutation might rather have a more direct effect on the interaction with host components, for example, by affecting a microtubule-binding interface of the protein or by affecting a specic fold in the protein that is required for microtubule association rather than for RNA binding. Intramolecular complementation of the P81S mutation by T104I and R167K mutations might suggest the stabilization of a fold by interaction of amino acids at positions 81, 104, and 167. Although our RNA-binding assays seem to exclude the possibility that the mutations affect the ability of MP to bind RNA per se, the mutations may still affect the quality of RNA binding. The rather diffuse RNA retardation pattern observed with the mutant proteins could indicate an attenuated ability of the mutant proteins to form a vRNP and therefore could provide a possible explanation for the low efciency of cell-to-cell spread shown by TMVMPP81S;T104I;R167K-GFP. It should be noted that in addition to the restoration of microtubule association, functional reversion was also correlated with the restoration of the ability of MP to accumulate to peripheral puncta and hair-like protrusions in protoplasts. These observations in protoplasts may be important and deserve further analysis. However, it is difcult as yet to draw conclusions with respect to their functional signicance, since corresponding structures were not detected in TMV-MPP81S;T104I;R167K-GFP-infected plant tissues. Finally, it should be emphasized that even though our experimental observations conrm the signicance of MP-interacting microtubules for the spread of infection, the particular role of this complex during infection remains to be elucidated. It has been proposed that microtubules support the spread of infection by serving as a track for the translocation of vRNA from replication sites to Pd (1, 14, 30, 31, 68). However, although this hypothesis is appealing, other potential mechanisms must be considered. For example, microtubules could support infection by transporting and anchoring ER-associated replication sites (42) or by mediating the storage, turnover, or degradation of MP. It is also conceivable that MP binds microtubules as a mechanism to interfere with a virus-induced plant defense response, e.g., RNA silencing (24). A precedent for such a potential role of MP is provided by one of the triple gene block MPs (25-kDa protein) of Potato virus X that functions as a virus-encoded silencing suppressor by interfering with either the production or trafcking of the non-cell-autonomous silencing signal (61).
ACKNOWLEDGMENTS V.B. and J.A.A. contributed equally to this work. We thank Vincent Brondani (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for invaluable help with RNA binding assays.
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