Interaction of DNA with the Movement Proteins of Geminiviruses Revisited

Geminiviruses manage the transport of their DNA within plantswith the help of three proteins, the coat protein (CP), thenuclear shuttle protein (NSP), and the movement protein (MP).The DNA-binding capabilities of CP, NSP, and MP of Abutilonmosaic virus (AbMV; family Geminiviridae; genus Begomovirus)were scrutinized using gel mobility shift assays and electronmicroscopy. CP and NSP revealed a sequence-independent affinityfor both double-stranded and single-stranded DNA, as has beenpreviously reported for other begomoviruses. MP interacted selectivelywith dimeric supercoiled plasmid DNA in the electrophoreticassay. Further apparent size- and form-selective binding capacitiesof MP have been previously reported for another geminivirus(Bean dwarf mosaic virus), but in the case of AbMV, they havebeen identified as the result of electrophoretic interferencerather than of complex formation. Without these complications,electron microscopy confirmed the assembly of double-strandedsupercoiled DNA with NSP and MP into conspicuous structuresand provided the first direct evidence for cooperative interactionof MP, NSP, and DNA. Based on these results and previous ones,a transport model of geminiviruses is discussed in which NSPpackages DNA and MP anchors this complex to the protoplasmicleaflets of plasma membranes and microsomes for cell-to-cellmovement.

INTRODUCTION

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Introduction

Plant viruses differ from animal viruses in their mechanismsof transport within the host. Once injected into a plant, theymove predominantly symplastically from cell to cell by usingphloem cells for long-distance transport. Viral movement proteinshave evolved to manage this task, especially to open the gates(plasmodesmata) between cells (27). What mechanism plant geminivirusesuse to mediate the transfer of their DNA from cell to cell isstill an open and challenging question. Most of our knowledgeabout this process is based upon indirect evidence, mainly derivedfrom in vitro assays, because the transport complex itself hasnot been identified during natural infection. Geminiviruses(36) package circular single-stranded DNA (ssDNA) into twinnedparticles, hence their name. Depending on the particular geminivirus,three different proteins are involved in mediating transportthrough host plants, the coat protein (CP), the nuclear shuttleprotein (NSP), and the movement protein (MP). These proteindesignations have been widely used in the literature (25) andwill be used here also, although in a strict sense all threeproteins (CP, NSP, and MP) may be called movement proteins,as will be discussed below.

Geminiviral capsids are composed of a single CP, encoded bythe gene V1, V2, or AV1 (also known as AR1), depending on thegeminivirus (56). The geminivirus genome may be segmented (bipartite)or not (monopartite), and in both cases one DNA molecule occupiesthe complete twinned particle (3, 56). For monopartite geminiviruses,CP is essential for systemic spread through the plant (5, 7,26, 28, 34, 52). For bipartite geminiviruses, CP is not absolutelynecessary for this task, and NSP (encoded by the gene BV1, alsoknown as BR1, on DNA B) substitutes for its function in transport(16, 18, 44, 46). However, if NSP is mutated, CP can still complementthis defect (20). Hence, CP and NSP share, in part, one redundantfunction and probably have a common evolutionary origin (R.Kikuno, H. Toh, H. Hayashida, and T. Miyata, Letter, Nature308:562, 1984). All geminiviruses express a further protein,designated MP, which is essential for viral movement from cellto cell. This protein is encoded by different genes, dependenton the virus: V1 or V2 for monopartite geminiviruses (4, 9,34, 52) and BC1 (also known as BL1) for bipartite geminiviruses(10, 11, 13, 23, 25, 30, 39, 48, 49).

Over the past decade, consensus has developed that MP cooperateswith NSP or CP during viral transport (15, 25). However, theviral DNA conformation which participates in the natural transportcomplex is unknown.

Geminiviruses replicate in cell nuclei and, consequently, haveto cross two borders during spread, the nuclear envelope andthe plasma membrane. Both CP and NSP are targeted to the nucleus,and at least NSP, hence its name, is able to shuttle betweenthe nucleus and the cytoplasm (19, 24, 33, 38, 46, 47, 50).Protein domains responsible for DNA binding and intracellularlocalization have been analyzed for several geminiviruses. Aconsensus exists that CP and NSP bind ssDNA as well as double-strandedDNA (dsDNA) in a sequence-independent manner (25).

By comparison, the role of MP in DNA binding and movement isstill controversial. Depending on the geminivirus analyzed,MP has been localized in "cell wall-enriched" (48) and microsomal(30) fractions as well as in the lumen of particular endoplasmicreticulum-derived tubules of the protophloem (51). More detailedinformation is available for the MP of Abutilon mosaic virus(AbMV). Cellular imaging of AbMV MP translationally fused togreen fluorescent protein or glutathione-S-transferase as reporterproteins showed that MP is targeted to the cell periphery inplants (55) or to microsomes and the plasma membranes in fissionyeast cells (1), respectively. Sequence analysis of AbMV MPrevealed no plausible domain which may function as a transmembranehelix. However, a membrane-binding domain of AbMV MP has beendelimited, and this domain harbors a putative amphipathic helixwhich may allow the insertion of MP into one leaflet of a membrane(54). It has been proposed that MP serves as a membrane anchorat the protoplasmic face of microsomes and plasma membranes,thus facilitating the movement of the transport complex alongthese membranes (54). Moreover, MP can shuttle between the nuclearenvelope and the cellular periphery in order to fulfill itstask, because green fluorescent protein-MP was also observedin the vicinity of nuclei in some cells (55).

Less is known about the viral DNA participating in the transportprocess. Microinjection experiments employing Bean dwarf mosaicvirus (BDMV) (29) provided evidence that NSP shuttles plasmidDNA between the nucleus and the cytoplasm and that MP transportsdsDNA, but no ssDNA, to the neighboring cell. Consistent withthese findings, Rojas et al. (35) have concluded from gel mobilityshift assays that ssDNA and dsDNA are bound in a form- and size-selectivemanner by NSP as well as by MP. Upon these findings, a "relayrace model" of transport was proposed in which NSP transfersviral dsDNA from the nucleus to the cytoplasm, from which itis delivered to MP for plasmodesmata crossing. Further supportfor DNA size selection during cell-to-cell transport has beenprovided by recent infection experiments (17).

A second model has been established which may be called the"couple-skating model." Applying DNA-cellulose chromatographyand protein overlay techniques for a distinct begomovirus (Squashleaf curl virus [SLCV]), Pascal et al. (31) observed strongerbinding of NSP to ssDNA than to dsDNA but only a weak interactionof MP with ssDNA and no interaction with dsDNA. These authorsfavor a transport model in which viral ssDNA is shuttled betweenthe nucleus and the cytoplasm in an NSP-containing complex,which then interacts with MP to move from cell to cell. Rojaset al. (35) tried to explain the differing results which ledto the relay race and the couple-skating models with the tissuetropism of the particular virus under investigation: whereasBDMV is able to spread into all leaf tissues, SLCV is phloemlimited. Alternatively, the apparent discrepancy in the interpretationsof the transport processes may have resulted from the differenttechniques used.

To resolve this debate, we initiated gel mobility shift experimentswith a phloem-limited begomovirus (AbMV) and followed the technicalapproach of Rojas et al. (35) as closely as possible. In general,we found the same results for AbMV as for BDMV, showing thatviral tissue tropism is not the essential factor. Scrutinizingthe experimental approach, however, led us to a different, morecautious interpretation which is closer to the couple-skatingmodel.

MATERIALS AND METHODS

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Materials and Methods

Protein expression and purification. AbMV proteins CP, NSP, and MP were expressed in Escherichiacoli BL21(DE3) and purified by differential centrifugation,urea washing, and sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis as described previously (53). Protein-containingbands were excised from the gels and electroeluted. Buffer exchangeagainst 0.2 M NaHCO₃ and 0.02% SDS (pH 8.1) and concentrationwas carried out by ultrafiltration (Centricon 10; Amicon/Millipore)(53), yielding stock solutions of 11 µg of CP/µl,3.5 µg of NSP/µl, and 4 µg of MP/µl.

Purification of dsDNA. Phagemid pBluescript II KS(+) or pBluescript-AbA 1.0V (full-lengthAbMV DNA A PstI clone [14] with the orientation to produce viralsense ssDNA from the F1 origin) was purified by cesium chloride-ethidiumbromide centrifugation from E. coli JM 83 according to standardprotocols (37). The plasmid was linearized with BamHI or theDNA A fragment was released with PstI and BglI before separationon 1% agarose gels and elution of the DNA with the GFX-Kit (Pharmacia).Sheared fish sperm DNA (molecular biology grade) was purchasedfrom Boehringer Mannheim. Marker DNA was obtained by digesting ${lambda}$ DNA with HindIII or with HindIII and EcoRI.

Preparation of ssDNA. pBluescript-AbA 1.0V was used to transform E. coli XL1-BlueMRF' (Stratagene) by using standard protocols (37). For replicationand packaging of ssDNA, helper phage R408 was inoculated witha multiplicity of infection of 10 according to the Promega guidelines(1996 protocols and applications guide, Promega, Madison, Wis.).Infected cells were grown under rotation (180 rpm) overnightat 37°C. This longer incubation time was used to accumulatemore recombined and deleted ssDNA molecules for the test system(37). Bacterial cells were pelleted by centrifugation (15 minat 12,000 x g at room temperature in a Sorvall centrifuge).The supernatant was digested by DNase I (2 U/ml) and RNase A(10 µg/ml) for 15 min at 37°C. Phages were precipitatedwith polyethylene glycol (5%), incubated on ice for 60 min,and centrifuged (15 min at 12,000 x g). The pellet was resuspendedin 400 µl of buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA)and extracted with phenol and chloroform. DNA was precipitatedwith ethanol and stored at –20°C.

Protein-DNA binding assays. Cytochrome c (from horse heart; catalog no. 9007-43-6; Sigma)was used for comparison. Binding buffer as described in reference35 (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 10% glycerol,1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) wassupplied with cytochrome c, CP, NSP, or MP and DNA in variousconcentrations as indicated in the figure legends. To this aim,protein stock solutions were diluted to 1 µg/µlwith binding buffer, mixed with DNA and further binding bufferto obtain the indicated concentrations in a reaction volumeof 20 µl, and incubated for 30 min at room temperature.The whole sample was loaded into gel slots, giving the indicatedconcentrations of proteins and nucleic acids per lane.

One- and two-dimensional gel electrophoresis. One-dimensional gel electrophoresis was performed in 0.7% agarosein 1x Tris-borate-EDTA containing 0.5 µg of ethidium bromide/ml(21). Samples were run at 5 V/cm for 1 to 2 h. The same gelsystem was used as the first dimension for two-dimensional analysisbut run at 0.5 V/cm for 22 h. For the second dimension, sampleswere run perpendicular to the former direction in 1.4% agarose,1x Tris-borate-EDTA, and 50 µg of chloroquine/ml withapplication of 45 V for 19 h. DNA was blotted onto Hybond N⁺membrane (Amersham) and hybridized with a digoxigenin-labeledpBluescript probe as described previously (21).

Western blotting using agarose gels. One-dimensional agarose gels were run as described above, resultswere documented by photography under UV illumination, and DNA-proteinwas blotted onto nitrocellulose membrane as for Southern transfers.Membranes were analyzed using antibodies against MP as describedpreviously (53).

DNA-protein complex spreading and electron microscopy. The spreading technique was performed as described in reference8. Ten microliters of in vitro binding assay mixture was dilutedwith 90 µl of binding buffer and supplemented with 2.5µl of 8% glutaraldehyde solution to fix the DNA-proteincomplexes for 10 min at 37°C. Ethidium bromide solution(12.5 µl; 1 mg/ml) was added, and 40-µl drops wereleft on Parafilm at room temperature for 10 min. Parlodium-carbon-coatedgrids were attached to the surface of these drops and to furtherdrops of water, uranyl acetate, and ethanol for 30 s each. Afterdrying at room temperature, evaporation from the grids was carriedout with 2 nm of platinum (1.6 kV/60 mA; 8° angle) and thegrids were analyzed by transmission electron microscopy as describedpreviously (21).

RESULTS

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Results

AbMV CP, NSP, and MP proteins were expressed in E. coli, purifiedusing differential sedimentation of inclusion bodies, limitedurea washing, SDS gel electrophoresis, and electroelution (53),and renatured by dilution in binding buffer as described inreference 35. These proteins, in various concentrations, wereincubated with different DNA samples of either viral or nonviralorigin and analyzed by gel mobility shift assays as describedin reference 35. Since CP and NSP are highly basic proteins,all assays included cytochrome c, which is known to bind DNAdue to its positive charge although it is not a proper DNA-bindingprotein. This additional control served to estimate the contributionof nonspecific interference with the electrophoretic behaviorof DNA in the particular gel system. The following results underscoredthe importance of this control and unraveled the narrow gapbetween true binding and electrophoretic interference.

For linear dsDNA of plasmid including full-length AbMV DNA A(Fig. 1a), for excised AbMV DNA A (Fig. 1b), for vector (Fig.1c), and for fish sperm DNA (Fig. 1e), as well as for undigestedplasmid DNA (Fig. 1d), CP (AV1) and NSP (BV1) shifted the DNAin a concentration-dependent manner. Remarkably, the shift wasmore pronounced for lower than for higher concentrations ofthe proteins. This is comprehensible if few bound protein moleculesinitially reduce the netto charge of an extended complex andDNA is then compacted into a smaller volume with increasingamounts of protein as expected for virus-like particles (42,43). The increasing bends of the bands with higher protein concentrationsindicate reconstructions of the complexes during electrophoreticmigration. In comparison, cytochrome c shifted the DNA but theretardation increased with larger amounts of protein (Fig. 1)until the DNA was ultimately moved to the cathode and was lostfrom the gel image. Interestingly, the migration behaviors ofmonomeric plasmid covalently closed circular DNA (cccDNA) (Fig.1d) and of fish sperm DNA with a size below 2 kb remained unaffectedby the viral proteins but not by cytochrome c. As for BDMV (35),AbMV CP and NSP bound dsDNA in a form- and size-selective mannerwithout any sequence preference.

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FIG. 1. Agarose gel analysis of the interaction of various DNA samples with AbMV CP (AV1), NSP (BV1), MP (BC1), and horse cytochrome c (Cytc). (a) Linearized pBluescript with inserted DNA A. (b) Linearized DNA A. (c) Linearized pBluescript. (d) Plasmid preparation of pBluescript. (e) Fish sperm DNA. Protein concentrations were 0, 1, 2, and 4 µg/lane, respectively, for the AbMV proteins and 0, 2.5, 5, and 7.5 µg/lane (a to d) or 0, 5, 10, and 20 µg/lane (e) for cytochrome c. Increasing concentrations are indicated by triangles. Conformations of DNA are indicated as linear (lin), open circular (oc), or covalently closed circular (ccc), and 1 and 2 indicate monomeric and dimeric forms, respectively. DNA concentrations were 50 ng/lane (a to c), 1.5 µg/lane (d), and 1 µg/lane (e). M, lambda HindIII fragments as size markers (in kilobase pairs). Asterisks indicate shifted bands under discussion.

A more subtle and different effect was found for AbMV MP (BC1).The migration behavior of linear dsDNA was hardly changed (Fig.1a to c). A slight transient shift may be inferred from Fig.1a, but this was not observed in all gels. Similarly, a smallwindow of low fluorescence was observed at the highest concentrationof MP with fish sperm DNA (Fig. 1e), and this window was qualifiedby the following experiments. In contrast, a transient concentration-dependentshift of certain DNA conformations was observed with undigestedplasmid DNA and MP (Fig. 1d) and was reproducible in severalgels. The fluorescence of the bands corresponding to dimericcccDNA (2cccDNA) or monomeric open circular DNA (1ocDNA) anddimeric open circular DNA (2ocDNA) was diffuse with 1 and 2µg of protein/lane but reappeared as sharp bands at thehighest concentrations. Although this result together with someinfluence of AbMV MP on the migration of fish sperm DNA (Fig.1e) may be interpreted to support DNA size selection of ABMVMP, further experiments described below showed that this conclusionis not reliable.

To monitor the interaction of the AbMV MP with ssDNA (Fig. 2),we used a helper phage to produce ssDNA from a construct ofpBluescript and AbMV DNA A in the viral sense. We took advantageof the fact that, upon longer multiplication of phages, recombinantand deleted DNA molecules accumulate (37). Plasmids with insertedDNA A were found to be more prone to such recombination anddeletion than the vector alone, possibly because the geminiviralplus strand origin of replication was active to a certain extentin bacteria, as has been reported for other geminiviruses (41).In consequence, a ladder of ssDNA, circular and linear forms,was produced. To make sure that only ssDNA was monitored inthis assay, phages were polished with DNase I before DNA releaseand the resulting preparation was checked by S1 nuclease digestion(data not shown). DNA corresponding to a major band comigratedwith viral monomeric or slightly smaller ssDNA (Fig. 2) as faras can be deduced from nondenaturing gels by comparison withdsDNA size standards (Fig. 2) and pBluescript ssDNA (data notshown). The DNA corresponding to this band consisted of plasmidand DNA A sequences since it hybridized with a pBluescript-specificprobe (Fig. 2) and DNA A (data not shown). The migration behaviorof this small DNA was not affected by CP, NSP, or MP (Fig. 2ato c). On the contrary, a window of retardation of DNA was exhibitedfor CP and NSP and, to a lesser extent, MP, showing the characteristicretardation and reacceleration of the DNA with increasing proteinconcentrations (Fig. 2).

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FIG. 2. Southern blot of helper phage-produced ssDNA (pBlue with inserted DNA A; 50 ng/lane) in the presence of AbMV CP (AV1) (a), NSP (BV1) (b), and MP (BC1) (c) with 0, 1, 2, 4, and 6 µg/lane, respectively, from left to right. Agarose gels were run as those described in the legend to Fig. 1, which did not allow an exact size reference with double-stranded markers, but the lowest band (1ss) comigrated roughly with monomeric ssDNA of AbMV. Hybridization was done with a pBluescript-specific probe. For comparison, part of the ethidium bromide-stained source gel with the fifth lane of panel c and ${lambda}$ markers (in kilobase pairs) is shown at the same magnification. Asterisks indicate shifted bands under discussion.

To summarize the results of electrophoretic assays with dsDNAand ssDNA, it appears that DNA molecules running ahead of the ${lambda}$ standard reference band of 2 kb are not affected in migrationby the viral proteins in contrast to cytochrome c. Whereas theretardation effect of AbMV CP and NSP is very prominent forall DNA partners, it is weak and transient for some plasmidforms, fish sperm DNA, and ssDNA with AbMV MP.

Because of the peculiar behavior of AbMV MP, its interactionwith plasmid DNA was analyzed in greater detail (Fig. 3). Atwo-dimensional gel was used to decide whether the MP-retardedDNA was predominantly 1ocDNA or 2cccDNA, which comigrate underthe chosen gel conditions. Samples in the absence and presenceof MP were run on a first agarose gel (as that in Fig. 1) butwith lower voltage to better resolve the two comigrating plasmidbands (Fig. 3a). Subsequently, the DNA was run perpendicularlyon a second gel in the presence of chloroquine, which intercalatesinto dsDNA and thereby resolves the topoisomers of cccDNA (Fig.3b and c). For a detailed description and interpretation oftwo-dimensional gels, see references 21 and 32. The double bandin the first dimension (Fig. 3a) was well separated in the seconddimension (Fig. 3b). Since the upper band exhibited a seriesof topoisomers and the lower band exhibited a typical oval spot,it can be concluded that the upper band results from dimericsupercoiled DNA and not from ocDNA.

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FIG. 3. One-dimensional (a) and two-dimensional (b and c) gel analysis of plasmid DNA (pBluescript; 1.8 µg) in the absence (–) or presence (+) of 1 µg of MP (BC1). M, size marker as shown in Fig. 1. One-dimensional gels were run as those described in the legend to Fig. 1 but with lower voltage and longer time, and two-dimensional gels were run with the same first dimension (1st) as in panel a and a second dimension (2nd) in the presence of 50 µg of chloroquine/ml to resolve the topoisomers of supercoiled DNA. DNA conformations are indicated as in Fig. 1. Note that the dimeric supercoiled form runs slightly more slowly than the open circular form in the first dimension (b) and that the migration of both forms is influenced by the presence of MP (c). 2xoc, dimeric open circular; 1ccc, monomeric covalently closed circular.

In most gels examined so far (Fig. 1d and 4, lane 7, and datanot shown), MP retarded 2cccDNA preferentially in comparisonwith 1ocDNA. In some gels, however (Fig. 3a and 4, lanes 8 to10), the migration of 1ocDNA was also changed. Moreover, MPselectively retarded additional plasmid DNA, presumably fromreplicative intermediates, in the center of the gel in Fig.3b. This retardation is represented by the smear between 2cccDNAand 1ocDNA forms as indicated in Fig. 3c. By comparison, theelectrophoretic behavior of 1cccDNA, 2ocDNA, and other multimericforms of plasmids was not changed in the presence of MP.

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FIG. 4. Comparison of the migration behaviors of plasmid DNA (pBluescript; ethidium bromide-stained agarose gel) (a) and MP protein (Western blot of the above-mentioned gel developed with anti-AbMV BC1 antibody) (b). Lanes 1 and 2: 1 µg of MP (BC1) was incubated in the presence of SDS at 65 and 95°C, respectively, before loading. Lanes 3 to 5: 1, 2, or 3 µg of MP (BC1) was loaded without plasmid DNA and SDS. Lanes 6 to 10: 0, 1, 1.5, 2, or 3 µg of MP (BC1), respectively, was combined with 1.8 µg of plasmid DNA. M, size marker as shown in Fig. 1. Brackets in lanes 7 are of the same size for comparison of the migration distance of the protein in panel b with that of the corresponding DNA in panel a. +, presence; –, absence. DNA conformations are indicated as in Fig. 1.

Before drawing final conclusions from the electrophoretic assays,we noticed that the sizes of the DNA retarded by MP varied withdifferent electrophoretic conditions. Thus, we suspected thatelectrophoretic interference might play a role and wanted toscrutinize this possible artifact. We reasoned that electrophoreticinterference would retard the movement of DNA but not necessarilythat of protein on account of the different charges and sizesof protein and DNA. On the other hand, a true complex shouldbe recognized by the comigration of protein and DNA. Therefore,MP was analyzed in the absence and presence of plasmid DNA onagarose gels as before but was additionally blotted onto nitrocelluloseto be detected by a specific antibody (Fig. 4). For comparison,SDS-denatured MP was run and blotted in parallel. Most importantly,MP migrated within the previously detected fluorescence window(Fig. 4b), irrespective of whether DNA was present or not. Thisfinding lends additional weight to the suspicion that the nonfluorescingwindow may be caused by electrophoretic interference. Otherwisethe protein should also be retarded and comigrate with the shiftedDNA. The only exception to this general observation was a minoramount of MP which appeared as a tiny protein band (Fig. 4b,lanes 7 and 8) comigrating with the position of 2cccDNA aheadof the free MP protein's front (compare Fig. 4a and b). Becausethe ethidium stain (Fig. 4a) is less sensitive than Westernblotting (Fig. 4b), the respective band of 2cccDNA is seen onlyin the Western blot but with the lowest protein concentrationsapplied (1 and 1.5 µg of protein versus 1.8 µg ofDNA) (Fig. 4b, lanes 7 and 8). Since no protein comigrated withocDNA, we attribute its retardation to electrophoretic interferencewhich may be indirectly enhanced by the formation of a complexof MP and 2cccDNA. With this explanation, it is comprehensiblewhy the results of the electrophoretic assays depended on thevoltage and the running time (compare Fig. 1d, 3a, and 4a; alsodata not shown).

To avoid the conflicts presented by electrophoretic interference,complex formation was visualized by electron microscopy (Fig.5). Plasmid DNA was incubated with NSP, MP, or both and spreaddirectly onto the drop surface (21). Without the addition ofcytochrome c as a spreading agent, as it is used in the classicalKleinschmidt technique (6), naked DNA is poorly resolved inthe electron microscope at an acceleration voltage of 60 kVcompared to 40 kV (21). Moreover, the tested proteins contributeto the background grains according to whether they are freeor bound to DNA. Under these conditions, samples with NSP andDNA exhibited mainly collapsed amorphous bodies and only a fewcircular DNA molecules (Fig. 5a). The presence of MP resultedin pronounced spreading of DNA with a relaxed appearance (Fig.5b). The combination of NSP and MP produced conspicuous beads-on-a-stringstructures of open extended rings harboring characteristic bubblesat one site of the complex (Fig. 5c and d). Such a type of structureis expected if supercoiled DNA was packaged by NSP and MP proteinsand concomitantly unwound so that part of the dsDNA had to bemolten to compensate for the topological stress.

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FIG. 5. Electron micrographs of plasmids (pBluescript) spread after incubation with AbMV NSP (a), MP (b), or NSP and MP (c and d). The samples were positively stained with uranyl acetate and shaded with platinum-carbon as described previously (21). Note the beads-on-a-string appearance of the complex in panels c and d. Typically the complexes open a bubble at one site (arrows). Bar represents 1 µm.

DISCUSSION

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Discussion

In summary, we conclude that size and form selection is probablefor CP and NSP of AbMV as shown for those of other geminivirusesbefore. In the case of AbMV MP, we can detect only a very lowlevel of form-specific binding to 2cccDNA. However, electronmicroscopy has provided the first evidence for formation ofcomplexes of NSP, MP, and DNA, consistent with the couple-skatingmodel. The results confirm the previously identified role ofCP and NSP as sequence-independent DNA-binding proteins (Fig.1 and 2) and resolve a discrepancy in the literature concerningthe DNA-binding capacity of MP (Fig. 3 and 4).

In contrast to the basic cytochrome c as a control for a nonspecificand biologically nonfunctional DNA-binding protein, CP and NSPdid not change the migration behavior of 1cccDNA of plasmids(Fig. 1d), of linear fish sperm dsDNA smaller than 2 kb (Fig.1e), and of monomeric ssDNA of viral genome size (Fig. 2). Similarresults were previously reported for BDMV and dsDNA and wereinterpreted as indicative of the DNA size selection capabilityof CP and NSP (35). In the case of ssDNA binding, Rojas et al.analyzed only M13 DNA as a binding partner. M13 DNA is morethan twice as large as geminivirus ssDNA, so a direct comparisonwith our results is impossible.

The in vitro DNA-binding results obtained for BDMV CP and NSPhave been reproduced here for the proteins of the phloem-limitedAbMV. We do not know whether the proposed size selection capabilityof NSP is biologically functional until the transport complexhas been isolated from infected plants. On the other hand, CPmust be able to package viral ssDNA of genomic and subgenomicsizes to form twinned particles or half-size solitary particles(12, 56). These ssDNA samples were obviously excluded from theprotein binding in the gel mobility shift assay (Fig. 2a). Severalexplanations could hold for the failure of CP to bind genome-sizessDNA. Either there is still some, until now undetected, sequence-specificCP binding, or CP protein may be posttranslationally modifiedin plant cells in order to fulfill its primary biological task.Finally, a gel mobility-based detection system may be inappropriatefor monitoring such an interaction on account of its electrophoreticproperties. DNA of higher velocity may be removed from complexesby electrical forces during the run in agarose. The last argumentis strengthened by the observation that all DNA samples, irrespectiveof their nature, which migrated faster than 2-kb ${lambda}$ standard fragmentsremained unaffected.

Sequence-independent formation of complexes of CP and NSP withlarger ssDNA and dsDNA is substantiated by the reversion ofelectrophoretic mobility with increasing protein concentrations.In an approach different from that of Rojas et al. (35), whoused His-tagged proteins in most experiments, we have employedproteins lacking fusion domains throughout the study. The results,however, were essentially the same, confirming that the proteinsthemselves, and not the fusion partner, were responsible forthe binding activity. For MP, we rely only upon a form-specifictransient interaction with 2cccDNA (Fig. 2d, 3, and 4). Thisinteraction was verified by the results of the reciprocal assay,the change in mobility of the protein (Fig. 4b). In contrast,all indications for size-selective DNA binding of MP have tobe judged with caution. Due to the coincidental migration behaviorof MP and DNA in the presumed size window, electrophoretic interferencecannot be excluded as the main cause for mobility changes.

A very low binding affinity of MP for ssDNA, but not for dsDNA,was found for SLCV by using chromatographic techniques (31).Interaction with 2cccDNA, as found in our study, is not necessarilyproof per se of dsDNA-binding capacity. Under topological stress,dsDNA may be molten to expose ssDNA stretches. Correspondingly,cccDNA becomes accessible for the ssDNA-specific S1 nucleasesas has been shown for geminiviruses (45). Interaction of MPwith such regions of dsDNA molecules may change the topologyof the circular DNA and thereby its mobility in gels (Fig. 1,3, and 4) as well as its appearance in electron micrographs(Fig. 5b to d). The presence of bubbles in the circular complexes(Fig. 5c and d) is the most suggestive representation of suchtype of interaction.

Until now, indirect evidence has suggested that begomovirusmovement proteins NSP and MP cooperate in cell-to-cell transport.Wild-type SLCV MP redirected NSP from the nucleus to the cellperiphery in protoplasts (38-40), whereas specific point mutationsin MP (39) or NSP (38) prevented this migration. Similarly,in plant tissues, AbMV MP redirected NSP to the cell peripheryand into the next cell's nucleus (55). Further neighbor cellsreceived NSP only if the NSP-reporter construct was coinoculatedwith fully infectious viral DNA. This result is up to now thestrongest available evidence in a biological context that NSPis transported intercellularly. The cooperation of MP and NSPwithin the cell seems to be independent of the host species,because the same redirection of NSP has been observed in fissionyeast upon ectopical expression of unfused AbMV MP and NSP (unpublisheddata). To study localization of MP at the cell periphery ingreater detail, freeze-fracture immunolabeling was carried outin yeast and showed an intimate association of this proteinwith the protoplasmic leaflets of microsomes and plasma membranes(1). The membrane-binding domain of AbMV MP has been delimited(54) and may serve as a membrane anchor for NSP or NSP-DNA complexes.Here, we have presented the first direct evidence of a physicalinteraction of MP, NSP, and DNA by using electron microscopy.The beads-on-a-string appearance of the complexes differs fromthat of viral chromatin as it has been visualized previously(2), but the regular organization of the structures in Fig.5c and 5d resembles linear structures, which were isolated frombegomovirus-infected Sida micrantha plants in the early daysof geminivirus research (22). The excellent spreading of theMP-NSP-DNA complexes for electron microscopy may be attributedto the hydrophobic portion of MP, which may orient the otherwisecollapsed NSP-DNA complex to the drop surface and provide improvedbinding to the mainly hydrophobic carbon layer of the grid film.Perhaps this property of MP also allows spreading of NSP-DNAcomplexes at the surface of cellular membranes, which couldbe suitable for threading of the complexes through small poressuch as plasmodesmata.

In conclusion, we prefer the couple-skating model of geminiviraltransport as described in reference 25 in which viral DNA (whethersingle or double stranded, circular or linear remains to beshown) is packaged by NSP, attached to membranes by MP, andtransported along the membranes through plasmodesmata to thenext cell.

ACKNOWLEDGMENTS

We thank C. Kocher and S. Mangold for skillful technical assistanceand J. Stanley and R. Ghosh for critical reading of the manuscriptand helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology and Plant Virology, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany. Phone: 49-711-685-5070. Fax: 49-711-85-5096. E-mail: holger.jeske{at}po.uni-stuttgart.de.

REFERENCES

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