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Journal of Virological Methods 166 (2010) 77–85
Contents lists available at ScienceDirect
Journal of Virological Methods
journa l homepage: www.e lsev ier .com/ locate / jv i romet
n vitro assembly of Tobacco mosaic virus coat protein variants derived fromssion yeast expression clones or plants
nna Mueller, Anan Kadri, Holger Jeske, Christina Wege ∗
niversity of Stuttgart, Institute of Biology, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, D-70550 Stuttgart, Germany
rticle history:eceived 7 December 2009ccepted 22 February 2010vailable online 26 February 2010
eywords:
a b s t r a c t
Tobacco mosaic virus (TMV) derivatives are explored currently extensively with regard to nanotechno-logical applications. Since certain technically desired TMV mutants may not be accessible from plants,the utility of the fission yeast Schizosaccharomyces pombe for heterologous production of TMV coat pro-tein (CP) variants was explored, including wild-type (wt) CP and two genetically engineered mutants:TMV-CP-His6 containing a C-terminal hexahistidine (His6) tag, and TMV-CP-E50Q with enhanced lateral
obacco mosaic virusission yeastchizosaccharomyces pombeelf-assemblyoat protein mutantsanotechnology
CP subunit interactions. After establishing expression clones and protocols for enrichment of the CP vari-ants, their ability to reconstitute TMV-like nanostructures in the presence or absence of RNA was testedin comparison with the corresponding plant-derived CP variants, which were expressed from infectiousTMV constructs. Both TMV-CP-E50Q and TMV-CP-wt yielded TMV-like rods, irrespective of the proteins’source. In contrast, His-tagged CP from plants produced only short rods in an inefficient manner, andno rods at all when expressed in yeast. This study introduces a novel approach to produce assembly
lso de
competent TMV CP, but a. Introduction
Tobacco mosaic virus (TMV), the type member of the genusobamovirus (Lewandowski, 2005), is a tube-shaped plus-strand (+)sRNA-containing plant virus with an average length of 300 nm, anuter diameter of 18 nm and an inner channel 4 nm in width. Itshysico-chemical properties have been characterized well, show-
ng an extraordinary stability in wide ranges of temperature andH (Klug, 1999; Mutombo et al., 1992; Nicolaieff and Lebeurier,979; Perham and Wilson, 1978; Wilson et al., 1976). The virion isomposed of about 2130 identical coat protein (CP) subunits whichre helically arranged on a single RNA component (Namba et al.,989; Sachse et al., 2007). Viral RNA and CP readily self-assemble
n vitro (Butler, 1999; Okada, 1986). These features in conjunctionith the high yield of TMV particles accessible from plants have
ed to an increasing interest to use TMV as a biotemplate buildinglock for nanotechnological applications (Balci et al., 2007, 2008;nez et al., 2003, 2004, 2006; Royston et al., 2008). The possibility
o manipulate its properties by genetic engineering has become an
ttractive approach to generate virions with enhanced chemoselec-ivity or novel reactivities on their surfaces and inside the channel.or instance, various TMV mutants were used for directed couplingeactions with large organic molecules on the inner or outer sur-∗ Corresponding author. Tel.: +49 0711 685 65073; fax: +49 0711 685 65096.E-mail address: [email protected] (C. Wege).
166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2010.02.026
monstrates its limitations.© 2010 Elsevier B.V. All rights reserved.
face of the virions (Demir and Stowell, 2002; Endo et al., 2006;Schlick et al., 2005; Yi et al., 2005), for the display of protein antigens(Bendahmane et al., 1999; Li et al., 2007; McCormick et al., 2006;Smith et al., 2006), or for the improved deposition of different metalspecies (Kadri, 2007; Lee et al., 2006; Royston et al., 2008). In the CPmutant E50Q, a glutamate residue is replaced by glutamine, whichled to reduced repulsive forces between adjacent CP subunits andenhanced the stability of virus-like rods (Culver et al., 1995). Themutant has attracted intensive research on tobamovirus disassem-bly and CP-mediated resistance (Bendahmane et al., 2007; Culveret al., 1995; Lu et al., 1996, 1998). When E50Q virus-like rods wereisolated from plants, they were often significantly longer than wild-type (wt) TMV (Bendahmane et al., 2007; Culver et al., 1995), andrarely contained RNA (Culver et al., 1995; Kadri, 2007). Hence, thisTMV variant might represent an interesting CP source for the pro-duction of self-assembling elongated proteinaceous tube structuresup to several micrometers length, and was therefore established asplant-infectious clone in our lab (Culver et al., 1995; Kadri, 2007). Inorder to functionalize the outer surface of TMV, a second CP mutantwas generated, which carries a hexahistidine tail (His-tag) at its C-terminus (Kadri, 2007). His-tagged biomolecules have been appliedas templates for several kinds of coupling reactions in nanotechno-
logical research (Banerjee et al., 2003; Chatterji et al., 2005; Slociket al., 2002).The ectopic expression of viral CP in a suitable heterologous sys-tem may constitute an appealing alternative to the work with plantsand infectious virus. For technical purposes, a routine production
7 ologic
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8 A. Mueller et al. / Journal of Vir
rocess for genetically tailored TMV CP monomers, which are capa-le to assemble in vitro, may also permit further degrees of freedom,llowing for changes of the amino acid sequence which otherwiseould conflict with viral multiplication and spread inside plants.
he CP of wt TMV has been expressed to high amounts in bac-erial cells (Endo et al., 2006; Hwang et al., 1994, 1998; Shire etl., 1990). However, the prokaryotically produced CP differed ints polymerization behaviour from plant-derived CP (Hwang et al.,994; Shire et al., 1990). Furthermore, in vitro reconstitution ofirus-like rods from RNA and the heterologously expressed CP wasot possible (Hwang et al., 1994), or only at a reduced rate (Shiret al., 1990). To circumvent these problems, the eukaryotic fis-ion yeast Schizosaccharomyces pombe has been tested recently forhe ectopic production of TMV CP, since in its molecular geneticroperties, this organism is related more closely to higher eukary-tes than baker’s yeast (Basi et al., 1993; Eckart and Bussineau,996; Forsburg, 1999; Forsburg and Rhind, 2006; Matsuyama etl., 2008; Maundrell, 1993). Indeed, fission yeast-expressed TMVt CP formed TMV-like rods spontaneously inside the cells (Kadri,
007). The usefulness of fission yeast was also shown for the pro-uction of other plant viral proteins in the course of experiments onhe DNA-containing geminiviruses (Aberle et al., 2002; Frischmutht al., 2007; Kittelmann et al., 2009; Kleinow et al., 2008, 2009).his study focuses on the ectopic production of the TMV CP vari-nts TMV-CP-His6 and TMV-CP-E50Q, and compares them with wtMV CP. The His-tagged CP was expressed in fission yeast andnriched by immobilized metal affinity chromatography (IMAC).he other TMV CP variants were received by a two-step procedureombining treatment at low pH followed by heating. The capabil-ty of such enriched proteins to assemble in vitro was tested in theresence or absence of in vitro transcribed RNA harboring the TMVequence of the origin of assembly (OAs). These results were com-ared to those with the corresponding TMV CP variants preparedrom plant-derived virions.
. Material and methods
.1. Construction of yeast expression clones
For fission yeast expression, constructs named TMV-CP-wt-fy,MV-CP-E50Q-fy and TMV-CP-His6-fy were designed using S. pombexpression vectors based on plasmid pESP1-pREP2 (Frischmuth etl., 2007). The vector carries a leucine auxotrophy marker, andarget genes are controlled by the thiamine-repressible nmt1 (no
essage with thiamine) promoter (Maundrell, 1993). The differentMV CP sequences were amplified from plant-infectious full-lengthMV cDNA clones, based on plasmid p843 pe35TMVr.1 (kindlyrovided by E. Maiß, Hannover, Germany; see Kadri, 2007 foronstruction of the mutants), by PCR using the primers speci-ed in Table S1. The resulting fragments were inserted into theamHI restriction site of pESP1-pREP2 according to standard proce-ures (Sambrook and Russell, 2001), and the recombinant plasmidsere propagated in Escherichia coli DH5� cells (New England Bio-
abs, NEB). The identity of the inserted sequences was verified byequencing using specific primers (Table S1).
.2. Heterologous expression of TMV CP variants in fission yeast
The leucine-auxotroph fission yeast strain SP-Q01 (Stratagene,at. # 200319) was transformed with the recombinant plasmids
rom E. coli following a small scale lithium chloride transforma-ion protocol (Forsburg and Rhind, 2006). Positive clones wereelected on Edinburgh minimal medium (EMM; Forsburg andhind, 2006) lacking leucine. Expression experiments were con-ucted as described by the supplier of the yeast strain (Stratagene).
al Methods 166 (2010) 77–85
Briefly, a single yeast colony was inoculated to 10 ml YES medium(Forsburg and Rhind, 2006) and grown for 18–19 h at 30 ◦C withshaking at 250 rpm. Culture growth was monitored by absorptionmeasurements at 600 nm (OD600) and counting cell numbers usinga Neubauer hemocytometer. These precultures were diluted into50 ml YES medium to yield an initial OD600 between 0.3 and 0.4and grown for additional 4–5 h (30 ◦C, 250 rpm). After two wash-ing steps with doubly demineralised water (ddH2O), cells wereresuspended in EMM (10–100 ml) to start expression at 0.1 OD600.For control cultures, the medium was supplemented with thi-amine (final concentration [f.c.] 5 �M) to repress the target gene.Cells were grown for up to 18 h (30 ◦C, 250 rpm) and harvested bycentrifugation. Cell pellets containing about 1.6 × 109 cells werewashed with phosphate buffered saline (PBS, pH 7.4; Sambrookand Russell, 2001) and stored at −80 ◦C.
2.3. Analysis of protein expression
Frozen cells from expression experiments were resuspendedin 500 �l PBS lysis buffer containing 0.2% Tween-20 (PBS-T, pH7.4) and phenylmethylsulfonyl fluoride (PMSF; f.c. 1 mM; from a200 mM stock solution in ethanol) and broken by adding one vol-ume of acid washed glass beads (Sigma; Cat. #G8772; 435–600 �mdiameter) per two volumes cell suspension and vigorous vor-texing for at least 2 × 5 min. The success of cell-rupture wasexamined by phase contrast light microscopy (Zeiss Axioskop).Lysate solutions were removed from the glass beads, and afterrinsing the beads again twice with 500 �l lysis buffer, the bufferfractions were pooled and the resulting crude extract separatedinto a supernatant (S) and a pellet (P) fraction by centrifugation(10 min, 10,000 × g, 4 ◦C). Pellets were resuspended in a volumeof lysis buffer corresponding to the volume of the supernatant,and aliquots (7–10 �l) were used for protein gel electrophore-sis (SDS, 15% polyacrylamide; SDS-PAGE) according to standardprocedures (Laemmli, 1970). Gels were stained with Serva violet-17 (SERVA Electrophoresis GmbH, Heidelberg) or by silver (Blumet al., 1987). For western blots, the proteins were transferred tonitrocellulose membranes (Protran BA85; Schleicher & Schuell;Dassel; Germany) by semi-dry electro-blotting (Towbin et al., 1979)and probed either with TMV-CP- specific rabbit IgG (580 �g/ml,diluted 1/1000; kindly provided by Prof. K.-W. Mundry, Stuttgart,Germany) and goat-anti-rabbit alkaline phosphatase (AP) con-jugate (goat-anti-rabbit-AP; Rockland, catalogue #611-1502) assecondary antibody, or with Penta-HistidineTM antibody frommouse (100 �g/ml; diluted 1/1000; QIAGEN catalogue #34660)in combination with AP-coupled goat-anti-mouse IgG (Rockland,catalogue # 1510-1602) according to the manufacturer’s protocol(QIAexpress® Detection and Assay Handbook, QIAGEN). AP activ-ity was detected using the chemoluminescence substrate CDP-Star(Roche Applied Sciences; Cat. # 12041677001). As a reference, wtTMV prepared from systemically infected plants was loaded inparallel with a standard protein ladder (Low Molecular WeightCalibration Kit for SDS Electrophoresis, GE Healthcare).
2.4. Enrichment of the TMV CP variants heterologously expressedin S. pombe
TMV-CP-His6-fy was enriched by aid of batch Ni-NTA agarosechromatography (QIAGEN; Cat # 30230). Supernatants of the crudecell extracts were supplemented with imidazole (f.c.: 5 mM) and1/4 vol. of Ni-NTA agarose. After shaking at 4 ◦C for 60–80 min, the
agarose beads were collected by centrifugation (1 min, 10,000 × g).The supernatant was removed yielding the remainder fraction (R)and beads were washed with the same volume of PBS-T contain-ing 20 mM imidazole (pH 7.4) as for the cell extract yielding thewash fraction (W). After pelleting of the Ni-NTA-agarose beads
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extracts were prepared and separated into a pellet (P) and a super-natant (S) fraction, both of which were analyzed by SDS PAGE andimmunodetection of the target proteins. Successful expression ofeach CP variant is shown in Fig. 1 for a representative experiment.
Fig. 1. Expression of TMV CP variants (CP-wt, CP-His6, CP-E50Q) in comparison tothe vector control (V) in S. pombe SP-Q01 liquid cultures. Fission yeast cell lysates
A. Mueller et al. / Journal of Vir
gain, proteins were eluted (E fraction) in PBS-T containing 250 mMmidazole (pH 7.4) with shaking for 5 min at room temperatureRT). If necessary, imidazole of the elution buffer was removedy ultrafiltration in Microcon® centrifugal filter units (Millipore,ermany; 10 kDa molecular weight cut-off). The final solution wasialyzed extensively against sodium potassium phosphate bufferSPP, 50 mM, pH 7.0, 4 ◦C) for 24 h.
For enrichment of TMV-CP-wt-fy and TMV-CP-E50Q-fy proteins,heir high stabilities towards pH changes and temperature werexploited. From the supernatants of the yeast crude extracts,00–500 �l were dialyzed (Slide-a-Lyzer mini dialysis units; Pierce;957.2) against 0.2 M sodium acetate buffer (pH 5.0) for 2 days at◦C, changing the buffer 2–3 times. Insoluble compounds were pel-
eted by centrifugation (10 min, 10,000 × g, 4 ◦C) and aliquots of theupernatant were incubated at 55 ◦C for 40 min followed by cen-rifugation (10 min, 10,000 × g). The supernatant of this preparationas used for further experiments. In order to achieve dissociation
f CP aggregates and hydrolysis of residual yeast RNA prior to initro assembly reactions with RNA, the acidic CP preparations wererst dialyzed against ddH2O (2–4 h, 4 ◦C) and subsequently against0 mM NaHCO3/Na2CO3 buffer (SCP; pH 10.2) for 48 h (Perham andilson, 1978). A final dialysis was performed against 50 mM SPP,
H 7.2 (24 h, RT).
.5. Preparation of TMV CP variants from plant-infectious TMVlones
Infectious cDNA clones (Kadri, 2007) TMV-CP-His6-pl andMV-CP-E50Q-pl were mechanically inoculated into Nicotiana ben-hamiana Domin plants in the five-to-six-leaf stage. An infectiousDNA clone for wt TMV was mechanically inoculated into Nico-iana tabacum cv Samsun nn in the five-to-six-leaf stage using�g of plasmid p843pe35TMVr.1 (E. Maiß, Hannover) per plant.fter 21 days, symptomatic leaves were harvested from systemi-ally infected leaves. wt TMV was prepared as described (Devasht al., 1981) with modifications (Kadri, 2007) and mutant TMVccording to Kadri (2007). Briefly, 1 g of frozen leaf material wasomogenized in a mortar in 10 ml SPP buffer (100 mM; pH 7.0,◦C), the sap was filtered through three layers of gauze yielding theltrate fractions (F). Centrifugation (5000 × g, 15 min, 18 ◦C) sepa-ated the filtrate into pellet 1 (P1) and supernatant 1 (S1). From1, virions of TMV mutant E50Q were precipitated with 8% (v/v)utanol and collected by centrifugation (5000 × g, 15 min, 18 ◦C).he resulting pellet (P2) was resuspended in a volume of buffer cor-esponding to the volume of the respective supernatant (S2). Fromirus-like rods, CP was extracted using an acetic acid-based methodFraenkel-Conrat, 1957). Briefly, one volume was mixed with twoolumes of glacial acetic acid and incubated on ice under occa-ional shaking for 20 min. After centrifugation (20,000 × g, 20 min,◦C), the SN was dialyzed against ddH2O for at least 2–3 days. Theeveloping precipitate was collected by centrifugation (20,000 × g,0 min, 4 ◦C), resuspended in half the starting volume of SPP buffer50 mM, pH 7.2) and incubated at RT for at least 24 h before insol-ble material was removed by a short low speed centrifugationtep (10,000 × g, 5 min, 20 ◦C). The final supernatant was used inelf-assembly experiments.
.6. In-vitro assembly of TMV CP with RNA
TMV CP solutions obtained from heterologous expression in fis-ion yeast or from plant-derived virions were tested for their ability
o build up TMV-like rods via in vitro assembly. RNA containing theMV origin of assembly (OAs) sequence was prepared by restrictionigestion of plasmid p843 pe35TMVr.1 with SnaBI and BsaBI (bothEB) followed by re-ligation of the resulting 6732 bp-fragment. Theenerated plasmid was named pTMV-B and contained a partial TMVal Methods 166 (2010) 77–85 79
genome (positions 2621–6395) downstream of a T3 promoter and20 nucleotides of the vector backbone. The plasmid was linearizedby BsiWI restriction to allow run-off transcription (MEGAscript®
T3 kit, Ambion). TMV CP proteins in SSP (50 mM, pH 7.2) weremixed with the transcripts in a weight ratio of 20:1 (CP:RNA) andincubated for 16–20 h at 30 ◦C (adapted from Butler, 1999 and ref-erences therein and Richards and Williams, 1972).
2.7. Transmission electron microscopy (TEM)
Immunosorbent electron microscopy (ISEM) was performed(Milne and Lesemann, 1984): Formvar-carbon coated copper grids(300 mesh; SCI) were dipped into ethanol (p.a.), air-dried, laidfor 5 min at RT on droplets (20 �l) of rabbit-anti-TMV IgG solu-tions (diluted 1/1000 in PBS buffer), rinsed with 10 drops of PBS,placed on 20–50 �l of the sample solution for 15–20 min at RT,rinsed with 10 drops of PBS, and placed on ddH2O for 20 s. Theenriched virus-like particles were negative-stained with 2% uranylacetate and analyzed in a TecnaiG2 Sphera (FEI) TEM at 120 kV.Digital images were taken and processed with the software ImageJ(http://rsbweb.nih.gov/ij/).
3. Results
3.1. Heterologous expression of TMV CP variants in fission yeast
The ORFs for wt TMV CP (to result in TMV-CP-wt-fy [-fy for fis-sion yeast-derived]), and CP variants which either harboured anamino acid exchange in their CP sequence (TMV-CP-E50Q-fy) or car-ried a C-terminal hexahistidine tag (TMV-CP-His6-fy) were clonedinto the expression shuttle vector pESP1-pREP2 and transformedinto the fission yeast strain S. pombe SP-Q01. The cell growth ofinduced, target protein-expressing cultures was not affected sig-nificantly by any of the constructs as compared to control cultureswhich harboured the vector plasmid only (V). Size and shape ofthe cells did not differ between the distinct clones, as analyzedby light microscopy (data not shown). After harvesting, crude cell
were prepared as described in 2.2. Aliquots of 9 �l from pellet (P) and supernatant (S)fractions were subjected to SDS PAGE (a) stained with Serva-violet, or to western blotimmunodetection (b) using anti-TMV-IgG. Arrows indicate the expected positionsof bands for TMV CP (CP-wt-fy and CP-E50Q-fy; lower arrow) and histidine-taggedCP (CP-His6-fy; upper arrow). As standards, 150 ng TMV from plants (T) and markerproteins (M) were applied with the indicated molecular weights (in kDa).
80 A. Mueller et al. / Journal of Virological Methods 166 (2010) 77–85
F in Fig.a
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of the other two variants precipitated during both acidification orheating (Fig. 4a/b, lanes #1 and #3). ISEM analysis of these fractionsrevealed long TMV-like rods as well (data not shown), indistin-guishable from the rods observed in the final enriched fractions.In conclusion, the acidification-based enrichment process was suc-
Fig. 3. Enrichment of TMV-CP-His6-fy from yeast by immobilized metal affinity chro-matography (IMAC). 200 �l of the lysate supernatant (LS) were incubated with 50 �lof Ni-NTA-agarose beads. After separating the beads, the “remainder” fraction (R)
ig. 2. Immunosorbent electron microscopy (ISEM) images of fractions describedntibodies. Scale bars: 200 nm.
or TMV-CP-wt-fy and TMV-CP-E50Q-fy, specific protein bands wereeadily identified in Serva-violet-stained gels (Fig. 1a). WhereasMV-CP-His6-fy was expressed to a lower amount (Fig. 1a), TMVP-specific antibodies recognized all single protein bands unequiv-cally at their expected positions (Fig. 1b). Owing to the histidineag, TMV-CP-His6-fy protein bands migrated more slowly than TMV-P-wt-fy, TMV-CP-E50Q-fy, and CP from a wt TMV prepared fromobacco plants as reference (T, Fig. 1). In all cases, similar amountsf heterologously expressed proteins were detectable in pelletsnd supernatants. From comparisons of band intensities on Servaiolet-stained polyacrylamide gels, the yields of target proteinsound in the supernatants of the cell lysates were estimated to bebout 0.2–0.4 mg TMV CP per litre culture volume.
The fractions were analyzed by transmission electronicroscopy using the enrichment of target particles by ISEM.
MV-like rods were visible in lysate fractions P and S fromMV-CP-E50Q-fy or TMV-CP-wt-fy samples, but neither from TMV-P-His6-fy nor from vector control samples (Fig. 2). The lengthistributions of the rods ranged from about 60 to 300 nm forMV-CP-wt-fy, or reached up to 600 nm for TMV-CP-E50Q-fy.
.2. Enrichment of the TMV-CP variants
Different procedures were tested to isolate heterologouslyxpressed TMV CP variants from yeast lysates. In the case of TMV-P-His6-fy, target proteins were enriched selectively by a batchrocedure using Ni-NTA agarose. SDS PAGE (Fig. 3a), and westernlot analysis with either anti-TMV IgG (Fig. 3b) or anti-penta-istidine-antibody (Fig. 3c) confirmed the identity of the targetrotein and the presence of the histidine tag in a single band,xcluding any deletions during the yeast culture. As visible on theilver-stained SDS gel (Fig. 3a), some contaminating yeast proteinsere still present in the final eluate. ISEM analysis of the enriched
ractions did not reveal any TMV-like rods or disks (data not shown).TMV-CP-E50Q-fy and TMV-CP-wt-fy proteins were selectively
nriched by subsequent acidification, heating, and precipitation ofenatured compounds. A representative result of this procedure ishown in Fig. 4. Compared to the respective lysates (Fig. 4a; lanes1), the target proteins were significantly delivered from yeast pro-
eins (Fig. 4a/b, lanes #4), yielding about 0.1 mg target protein peritre expression culture. ISEM analysis revealed long TMV-like rodsn these enriched fractions (Fig. 4c) of approximately 100–500 nmor TMV-CP-wt-fy, and 400 nm to 1 �m for TMV-CP-E50Q-fy. TMV-ike rods were never observed in lysates of vector control cells
1, following uranyl acetate negative staining. Anti-TMV-IgG were used as capture
that were processed in parallel (Fig. 4c, V). TMV-CP-His6-y was notenriched by this procedure, but completely precipitated after acid-ification (Fig. 4a/b lane #1). The sedimented material did not showany defined structure (Fig. 4c). In contrast, only a minor proportion
was collected, the beads were washed with 200 �l washing buffer, yielding the“wash” fraction (W), and eluted with 200 �l elution buffer (E). 7 �l of each solu-tion were separated on SDS PAGE and proteins were visualized by silver staining(a), or after western blotting detected by immunolabelling either with anti-TMV-IgG (b) or anti-penta-His-IgG (c). Standards M and T were as described in Fig. 1.Arrow indicates the position of the expected TMV-CP-His6-fy band.
A. Mueller et al. / Journal of Virological Methods 166 (2010) 77–85 81
Fig. 4. Enrichment of TMV-CP-wt-fy and TMV-CP-E50Q-fy by acidification and heating steps, in comparison to TMV-CP-His6-fy which is lost during these treatments, andv atantso ts aftea llets ws . 1). B
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ector control (V). The stepwise fractionation of the indicated yeast lysate supernf the western blot with anti-TMV-IgG (b), and ISEM as described in Fig. 2(c). Pellefter heating (lanes #3); supernatants after heating (lanes #4) are compared. The peupernatant. Arrows point at the expected positions of the target proteins (as in Fig
essful for obtaining TMV-CP-E50Q-y and TMV-CP-wt-y from yeastell lysates in a state amenable for assembly.
.3. Assembly of enriched TMV CP variants with RNA
Enriched proteins (Figs. 3 and 4) were dialyzed against a bufferuitable for in vitro assembly with RNA (see Section 2.4). An initro transcribed RNA of 3651 nts harbouring the TMV origin ofssembly was added (Section 3.4), expecting to build up rods withbout 170 nm in length.
TMV-CP-E50Q-fy and TMV-CP-wt-fy protein samples of enrichedractions (see above) were first dialyzed against carbonate bufferSCP, pH 10.2, see Section 2.4) in order to promote the dissociationf CP aggregates (Durham et al., 1971; Durham and Klug, 1971;erham and Wilson, 1978) and to remove any residual yeast RNA.SEM analysis confirmed that this treatment was effective in bothases (Fig. 5a and d). For in vitro-assembly with RNA, the proteinsere dialyzed against a buffer (50 mM SPP, pH 7.2; see Sections 2.4
nd 2.6) supporting assembly, and RNA was added. After a suffi-ient incubation time (24 h), preparations were analyzed by ISEMFig. 5b,c,e and f) and TMV-like rods re-appeared with both CP vari-nts (Fig. 5b and e). Only a few TMV-CP-wt-fy rods showed thexpected length (Fig. 5b), whereas the vast majority were muchonger (between 300 and 600 nm), indicating either head-to-tailssociation of monomeric units or the assembly of protein aloneFig. 5e; no example shown for TMV-CP-wt-fy), since control reac-ions without RNA showed long rods for both the two CP speciess well (Fig. 5c and f). TMV-CP-His6-fy did not yield any TMV-likeanorods at all.
.4. Self-assembly experiments with TMV CP variants purifiedrom plants
In contrast to the failure of TMV-CP-His6-fy protein to assemblen the previous experiments, TMV mutants carrying the same His-xtension of their CP were able to spread systemically and builtp virus-like rods in plants (Kadri, 2007). Thus, we investigatedhether the modified plant-derived CP would be able to assem-
le in vitro under the chosen conditions. His-tagged virus-like rods
rom TMV-CP-His6-pl (-pl for -plant-derived) were enriched fromystemically infected N. benthamiana plants, in parallel with TMVutant E50Q (TMV-CP-E50Q-pl) and wild-type virions (see Sec-ion 2.5). The different types of TMV-like particles accumulated inistinct fractions, as expected from previous experiments (Kadri,
was monitored by SDS PAGE separation and silver staining (a); immunolabellingr acidification (lanes #1); supernatants after acidification (lanes #2); precipitatesere resuspended in the same volume of SPP [50 mM; pH 7.0] as the corresponding
ars = 200 nm.
2007): TMV-CP-His6-pl in fraction P1 and TMV-CP-E50Q-pl in S1 or,after butanol precipitation, in P2 (Fig. 6a). In the case of TMV-CP-E50Q-pl, fraction S1 was chosen for CP preparation, because it gavehigher CP yields than P2 (data not shown, and Kadri, 2007).
Although the CP variants behaved substantially differently dur-ing purification (see Kadri, 2007 and Fig. 6a), they showed similarproperties during CP preparation. As described originally for wt CP(Fraenkel-Conrat, 1957), both CP mutants remained in the super-natant after treatment with acetic acid (Fig. 6b, F2) and precipitatedafter extensive dialysis against ddH2O (Fig. 6b, F3). For wt TMV,the protein yield available for assembly experiments was about50–60% of the original material, as estimated from SDS PAGE anal-ysis (not shown). For TMV-CP-His6-pl and TMV-CP-E50Q-pl, yieldswere somewhat lower (about 30–40% of the original material;Fig. 6b; F3 compared to F1).
For subsequent assembly, aliquots containing CP were eithersupplemented with the in vitro transcribed RNA containing theTMV OAs, or buffer in control reactions. As analyzed by ISEM (Fig. 7),the formation of TMV-like rods from TMV-CP-wt-pl was promotedby the addition of RNA to the samples (Fig. 7; compare CP-wt-pl -RNA and CP-wt-pl + RNA). Furthermore, the length of the rods wasgoverned by the length of the RNA scaffold. Similarly, no TMV-likerods were found when RNA was omitted from the reaction withTMV-CP-His6-pl (Fig 7; CP-His6-pl - RNA), but with RNA rods wereat least observed sporadically (Fig. 7; CP-His6-pl + RNA). Their lengthwas usually below the expected value of 170 nm. These structuresshowed a strong tendency to aggregate and 20S disk-like structureswere observed, irrespective of whether RNA was added or not (insetin Fig. 7).
TMV-CP-E50Q-pl proteins (Fig. 7; CP-E50Q-pl - RNA and CP-E50Q-pl + RNA) behaved similar to TMV-CP-E50Q-fy (compare Fig. 5e andf): long TMV-like rods were formed, independent of the presenceor absence of RNA. Their length (up to 1.5 �m observed) was notgoverned by the added RNA, as expected for this mutant.
4. Discussion
Three TMV CP variants, TMV-CP-wt, TMV-CP-E50Q, and TMV-CP-His6, have been successfully expressed in fission yeast, enriched
by adapted protocols, and tested for their in vitro assembly com-petence in comparison to the corresponding plant-derived CPvariants. To our knowledge, this is the first study on the suitabil-ity of a yeast-based synthesis of tobamoviral genetically modifiedcoat proteins. TMV-CP-wt-fy and TMV-CP-E50Q-fy, in contrast to
82 A. Mueller et al. / Journal of Virological Methods 166 (2010) 77–85
F ampled sence
TlhaUCpCawrisel
ig. 5. ISEM analysis of disassembled and subsequently reassembled yeast-derived sue to high pH (a and d), or subsequent re-assembly in the presence (b and e) or ab
MV-CP-His6-fy, spontaneously formed TMV-like rods of variableength within the yeast cells (Fig. 2), showing their ability to formomopolymeric nanotubes under ectopic conditions. All CP vari-nts were successfully enriched by different, adapted procedures.pon acidification and heating of yeast lysates containing TMV-P-wt-fy or TMV-CP-E50Q-fy, respectively, elongated TMV-like rodsolymerized (Fig. 4c), as already described for functional TMV wtP (Durham and Finch, 1972; Durham et al., 1971; Mutombo etl., 1992). The length of the particles ranged from 100 to 500 nmith wt, and up to 1 �m with E50Q CP. The formation of extended
ods is especially typical for the E50Q mutant, because the mutationn the lateral interaction face of the CP subunits reduces the repul-ive forces of two carboxylates between adjacent CP monomers (Lut al., 1996, 1998). In planta synthesized proteins exhibited simi-ar structures (Fig. 7, and Kadri, 2007). They were also found in
s of TMV-CP-wt-fy (a–c) and TMV-CP-E50Q-fy (d-f). Samples after initial disassembly(c and f) of RNA. ISEM was performed as described in Fig. 4 (Bars = 200 nm).
pellets and supernatants from yeast lysates after acidification andheating (data not shown). When enriched yeast-derived TMV-CP-E50Q-fy and TMV-CP-wt-fy proteins were depolymerized at highpH, and subsequently tested for assembly with RNA, TMV-like rodswere again efficiently formed. Their length, however, exceededfrequently the maximum aspect ratio expected for the scaffold-ing RNA. For the E50Q proteins, the results agree with the alreadyreported RNA-independent self-assembly of these proteins lead-ing to proteinaceous tubes completely or mainly devoid of RNA(Bendahmane et al., 2007; Culver et al., 1995; Kadri, 2007; Lu et al.,
1996). For TMV-CP-wt-fy, extended rods may be formed by head-to-tail association of RNA-encapsidating particles as well as by growthof additional protein tube portions, depending on the protein con-centrations in relation to RNA. In summary, the described similarbehaviour of both wt CP and E50Q CP from plants, and their respec-
A. Mueller et al. / Journal of Virological Methods 166 (2010) 77–85 83
Fig. 6. Enrichment of TMV CP mutants TMV-CP-His6-pl and TMV-CP-E50Q-pl from systemically infected plants, in comparison to fractions from mock-inoculated plants.M n 2.5F sed tom ctions#
tenoci
btwwTbmeWeiBWXm
Fvm
utant virions were enriched by differential centrifugation as described in Sectioractions P1 for TMV-CP-His6-pl and SN1 for TMV-CP-E50Q-pl were further procesaterial, fractions #2 (F2) the pellets obtained after treatment with acetic acid; fra4 (F4) the supernatants after this dialysis.
ive counterparts from yeast cells confirms the suitability of thectopic expression host for the production of these proteins foranotechnology applications. The quantities of the target proteinsbtained in yeast need further optimization, but they are suffi-ient for the small amounts used for nanotechnology experimentsn general.
TMV-CP-His6-fy was easily enriched from yeast crude extractsy affinity chromatography (Fig. 3) proving the functionality ofhe His-tag in binding nickel. However, the modified proteinas recalcitrant during assembly experiments. Histidine tags areidely used in protein purification (Arnold and Haymore, 1991;
erpe, 2003 and references therein), and only few problems haveeen reported. Some proteins showed reduced solubility or enzy-atic activity compared to their non-tagged counterparts (Busso
t al., 2003; Mohanty and Wiener, 2004; Pietzsch et al., 2000;oestenenk et al., 2004), failed to form oligomers (Amor-Mahjoub
t al., 2006; Halliwell et al., 2001; Wu and Filutowicz, 1999), or
mpaired their DNA binding properties (Amor-Mahjoub et al., 2006;uning et al., 1996; Halliwell et al., 2001; Shibagaki et al., 1997;u and Filutowicz, 1999). Heterologously expressed Potato virus(PVX) CP fused to an N-terminal His-tag formed virus-like fila-ents only after removal of the tag (Zayakina et al., 2009). On theig. 7. ISEM analysis of in vitro assembly of purified TMV-CP proteins which were prepaariants were incubated without RNA (−RNA) or with TMV OAs-containing RNA (+RNA)agnification. Bars: 200 nm, except for inset = 100 nm.
and their proteins separated on SDS-PAGE followed by Serva-violet staining (a).purify CP protein (b). Fractions #1 (F1) show aliquots of the respective starting#3 (F3) the pellets after dialysis of the supernatants against ddH2O, and fractions
other hand, successful expression and assembly of His-tagged viralCP has been reported (Chatterji et al., 2005; Maree et al., 2006) foricosahedral viruses which may have more degrees of freedom forsurface-exposed peptides. Bendahmane et al. (1999) have proposedthat the isoelectric point of epitopes at the surface of TMV is impor-tant and therefore positively charged residues are not toleratedunless they are compensated by negative charges. This conclusionmay explain the reduced stability of His-tagged CP rods or disksobserved, although also longer rods with packaged viral RNA wereobtained from plants (Kadri, 2007). Alternatively, the increasedtendency to aggregate may have obscured the detection of mul-timerized units by negative staining and electron microscopy.Aggregation of His-tagged proteins has been described as a com-mon problem (Renzi et al., 2006). In order to evaluate whetherthe chosen assembly conditions were suitable, the ability of plant-derived His-tagged CP (TMV-CP-His6-pl) was compared with itsyeast homologue, in parallel with the other plant-derived TMV CP
variants (TMV-CP-wt-pl and TMV-CP-E50Q-pl). Although the His-tagged virions precipitated in sodium phosphate buffer pH 7.0during differential centrifugation (Fig. 6a), their CP was solubilizedby acetic acid treatment and thus recovered in the same frac-tions as for TMV-CP-wt-pl and TMV-CP-E50Q-pl (Fig. 6b). Whereasred from systemically infected plants as described in Fig. 6. The indicated proteinunder conditions that favour assembly. Inset shows disk-like aggregates at higher
8 ologic
TtCbtaptfaefpHvacPitagirGCdcaobW
A
twtpLN
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A
A
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4 A. Mueller et al. / Journal of Vir
MV-CP-wt-pl formed virus-like rods with a length correspondingo the size of the scaffold RNA (Fig. 7; TMV-CP-wt-pl+RNA), TMV-P-E50Q-pl formed the extremely long TMV-like rods describedefore (Fig. 7; TMV-CP-E50Q-pl +/− RNA), and TMV-CP-His6-pl pro-eins yielded some virus-like rods with reduced size and disks afterddition of RNA (Fig. 7; TMV-CP-His6-pl + RNA). The majority of theroducts consisted of short 20 to 100 nm long rodlets. The observa-ion that hexahistidine-coated TMV particles were more efficientlyormed in N. benthamiana plants than during in vitro assembly,nd since mutations back to wt CP in these plants were carefullyxcluded (Kadri, 2007), the result may hint at mechanistic dif-erences between the assembly processes in vitro/in fungi and inlanta. Plant chaperones may prevent unspecific aggregation of theis-tagged proteins, thus enabling more efficient self-assembly ofirus-like rods (Boston et al., 1996 and references therein; Mayernd Bukau, 2005). For several viruses, the relevance of host cellhaperones for capsid assembly has been established (Sullivan andipas, 2001). For TMV CP mutants it was shown that misfold-ng may activate the ubiquitin/proteasome pathway in plants, andhus result in their ubiquitylation (Hamacher et al., 2003; Jockuschnd Wiegand, 2003; Jockusch et al., 2001). Following its heterolo-ous expression in E. coli, wt TMV CP did not multimerize on RNAn vitro; however, some progress in the formation of virus-likeods was achieved by a concomitant overexpression of bacterialroEL-GroES chaperones (Hwang et al., 1994; Shire et al., 1990).orrespondingly, also in vivo binding of TMV CP by GroEL wasemonstrated (Hwang et al., 1998). Although wtTMV is the biologi-al prototype for the capacity of polymers to self-assemble withoutssistance of chaperones, mutational variants may need chaper-ne function, if the chance for their misfolding is enhanced, as haseen shown for temperature-sensitive TMV mutants (Jockusch andiegand, 2003).
cknowledgements
We like to thank Cornelia Kocher for skilful technical assis-ance, and the gardeners Annika Allinger and Diether Gotthardt,ho took great care of our plants. We are very much obliged
o Prof. Nussberger and PD Dr. Michael Schweikert for sup-ort with the TEM facilities. The project work was funded byandesstiftung Baden-Württemberg (Kompetenznetz Funktionelleanostrukturen, KFNII TPC5) and subsidized by the DFG (SPP1165).
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.jviromet.2010.02.026.
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