Virus Research 145 (2009) 293–299

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Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV),P26 and P9, are self-interacting

Lucy R. Stewart, Min Sook Hwang, Bryce W. Falk ∗

Dept. of Plant Pathology, University of California, Davis, One Shields Ave., Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 29 April 2009Received in revised form 7 July 2009Accepted 28 July 2009Available online 7 August 2009

Keywords:ClosteroviridaeCrinivirusYeast-two-hybrid

a b s t r a c t

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid(Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise com-binations. Interaction was detected only for the P26–P26 combination. P26 self-interaction domains weremapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the crinivirusesBeet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus(LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologousproteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactionswith the six aforementioned LIYV proteins and each other. No interactions were detected for P5, butP9–P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members

Plasmalemma depositsLIYV

of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 haspreviously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs),

for P9

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. Introduction

Members of the genus Crinivirus in the family Closteroviridae arehitefly transmitted (+) ssRNA viruses emerging worldwide, withbroad range of agricultural and weedy plant hosts (Wintermantel,004; Wisler et al., 1998). Lettuce infectious yellows virus (LIYV)

s the type member of the genus and one of the most stud-ed criniviruses to date. Since the emergence of LIYV in thenited States in the early 1980s, other criniviruses transmittedy various whitefly vectors (Bemisia and Trialuerodes spp.) haveontinued to increase in distribution and importance worldwideCosta, 1976; Polston and Anderson, 1997; Wintermantel, 2004).ome criniviruses of current economic importance include Beetseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virusCYSDV), Lettuce chlorosis virus (LCV) Tomato chlorosis virus (ToCV),weet potato chlorotic stunt virus (SPCSV), Tomato infectious chloro-is virus (TICV), Potato yellow vein virus (PYVV), and Bean yellowisorder virus (BnYDV). Although there is variability in nucleotide

equences and the presence or absence of some open readingrames (ORFs), the criniviruses sequenced so far have genome orga-izations and gene complements similar to that of LIYV (Klaassent al., 1995), and study of LIYV has provided insight into Crinivirus

∗ Corresponding author. Tel.: +1 530 752 5218; fax: +1 530 752 5674.E-mail addresses: lrstewart@ucdavis.edu (L.R. Stewart), mshwang@ucdavis.edu

M.S. Hwang), bwfalk@ucdavis.edu (B.W. Falk).

168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2009.07.021

.© 2009 Elsevier B.V. All rights reserved.

features. Here we used a yeast-two-hybrid system to study interac-tion properties of LIYV-encoded proteins as well as some crinivirusorthologs.

Like LIYV, most members of the genus Crinivirus have bipar-tite genomes (except for Potato yellow vein virus, PYVV, for whichevidence of a tripartite genome has been reported; Livieratos et al.,2004). LIYV RNA1 encodes replicase proteins, with ORF1a predictedto encode a protein with protease, methyltransferase, and helicaseactivities and ORF1b predicted to encode the viral RNA-dependentRNA polymerase (Klaassen et al., 1995). All of the sequencedcriniviruses share similar organization of these ORFs. RNA1 3′ ORFsare quite variable among the criniviruses and even among isolatesof the same species (Cuellar et al., 2008; Tzanetakis and Martin,2004). In LIYV, RNA1 contains one 3′ ORF which expresses a 34-kDa protein, P34, from an abundant subgenomic RNA (Klaassenet al., 1995; Yeh et al., 2000). LIYV P34 functions by an unknownmechanism to enhance replication of RNA2, but not RNA1 (Klaassenet al., 1995; Yeh et al., 2000). LIYV RNA1 replicates efficiently inprotoplasts without RNA2 (Yeh et al., 2000).

LIYV RNA2 is predicted to encode seven proteins: P5, Hsp70h,P59, P9, CP, CPm, and P26. Biological functions of the RNA2-encodedproteins have so far been challenging to ascertain, since they are

unessential for replication processes readily observed in inoculatedprotoplasts (Yeh et al., 2000). Whole plant infection of this whiteflytransmitted, phloem-limited virus has been difficult to achieve formutant genotypes derived from infectious LIYV cDNA (Ng and Falk,2006), and no reverse-genetics systems have yet been developed

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94 L.R. Stewart et al. / Virus

or any other crinivirus. There are, however, indications of the rolesf at least some of the RNA2-encoded proteins. Five of these pro-eins, P5, Hsp70h, P59, CP, and CPm, are encoded by a gene blockonserved in the genus Closteroviridae called the “hallmark clos-erovirus gene array.” P5 is a predicted small hydrophobic proteinith a transmembrane helix (Klaassen et al., 1995). Hsp70h (ansp70 homolog), P59, CP (the major coat protein), and CPm (theinor coat protein) are virion components (Tian et al., 1999). LIYV

irions are flexuous rods with polar arrangement of the virion pro-eins: the viral RNA is encapsidated primarily by CP, with the CPmnd presumably the other virion proteins at “rattlesnake tails” as isharacteristic of the family Closteroviridae (Agranovsky et al., 1995;atyanarayana et al., 2004; Tian et al., 1999). In addition to the fiveonserved proteins encoded by the “hallmark array” genes, LIYVNA2 encodes two other proteins, P9 and P26. P9 is predicted to bencoded by a small ORF between the P59- and CP-encoding ORFs. A9-like protein is predicted to be encoded by a similarly positionedRF in all of the members of the genus Crinivirus sequenced so far

Dolja et al., 2006), but no function has yet been assigned to thisrotein. Finally, LIYV and all other criniviruses sequenced to datencode a protein of approximately 26 kDa (P26 for LIYV) from a 3′

NA2 ORF. LIYV P26 expression induces a unique LIYV cytopathol-gy, plasmalemma deposits (PLDs) (Stewart et al., 2009) which hasot been observed for other criniviruses studied to date (Medinat al., 2003). Other RNA2 ORFs predicted to encode small proteinsre variable in their presence, sequence, and position in the vari-us sequenced Crinivirus genomes (Aguilar et al., 2003; Kreuze etl., 2002; Tzanetakis and Martin, 2004; Wintermantel et al., 2005).

The consistent association of PLDs with LIYV virus-like parti-les (Medina et al., 2003, 2005; Pinto et al., 1988) prompted uso examine whether P26 might interact with virion components,erhaps in a polar manner. In addition, assessment of protein inter-ctions using a yeast-two-hybrid system is part of ongoing studieso better understand the functions and modes of action of the var-ous LIYV-encoded proteins. We tested LIYV proteins including theNA1-encoded P34 and RNA2-encoded P5, Hsp70h, P59, P9, CP,Pm, and P26. We also tested P26 orthologs from other criniviruses

or self-interaction properties. Our results provide novel informa-ion about two Crinivirus-specific proteins, P26 and P9.

. Materials and methods

.1. Cloning LIYV and Crinivirus genes into yeast plasmids

Genes were amplified by polymerase chain reaction (PCR) usingfu-Turbo polymerase (Stratagene, La Jolla, CA) according to theanufacturer’s instructions (see Table S1 for primer sequences).

IYV genes were amplified from RNA1 clone pSP9/55 or RNA2lone pSP6 (Yeh et al., 2000). BPYV and CYSDV P26 sequences werebtained as described previously (Stewart et al., 2009). LCV P26DNA was obtained from total RNA of LCV-infected plant tissueindly provided by Drs. James Ng and Nida Salem (UC Riverside)y reverse transcription with Superscript II reverse transcriptaseInvitrogen Corp., Carslbad, CA). The ToCV p27 gene was kindly pro-ided by Dr. Jesus Navas-Castillo (Estación Experimental La Mayora,álaga, Spain). PCR products were digested with NdeI/BamHI (for

5, Hsp70h, P59, P9, CP, LIYV P26, BPYV P26, CYSDV P26, andCV P26) or with NdeI/EcoRI (for P34 and CPm) and ligated intohese sites of pGADT7 and pGBKT7 vectors containing upstreamAL4 activation and binding domain sequences, respectively (Clon-

ech Laboratories, Inc., Mountain View, CA). LIYV P26 truncationsere ligated into NdeI/BamHI-cut pGADT7. The ToCV p27 gene was

loned into NdeI/XhoI sites of pGADT7, from which it was subclonednto pGBKT7 using NdeI/PstI. Ligation clones were sequenced andound to contain some consistent nucleotide changes compared to

rch 145 (2009) 293–299

published or provided sequences (Table S2). Constructs cloned intopGADT7 are denoted with an ‘A’ prefix (e.g. AP34); those cloned intopGBKT7 are denoted by a ‘B’ prefix (e.g. BP34).

2.2. Yeast-two hybrid assays

Small-scale lithium acetate-mediated transformation of yeaststrain AH109 was used for yeast-two-hybrid screening accord-ing to the manufacturer’s instructions (Matchmaker Two-HybridSystem 3; Clontech Laboratories, Inc., Mountain View, CA) exceptthat yeast incubation steps were carried out at 28 ◦C. Cotrans-formations were performed using 200 ng of each plasmid. For P5and P9 assays, 200 ng of bait plasmid and 500 ng of prey plas-mid mixture containing equal concentrations of each prey plasmidwere used. Transformed yeast cells were plated on agar mediaprepared as recommended by the manufacturer with various selec-tion stringencies: SD/-Leu/-Trp for low-stringency selection (L),SD/-Leu/-Trp/-His for medium-stringency selection (M), and SD/-Leu/-Trp/-His/-Ade for high-stringency selection. 3-Aminotriazole(3-AT) was used at 25 mM and 50 mM concentrations in medium-and high-stringency media, respectively. Protein interaction wasdetermined by colony growth on selective media and filter liftassays for �-galactosidase activity according to the manufacturer’sinstructions. Over 300 colonies in at least three experimental repli-cates were tested for each plasmid set, with blue color developmentafter 1–4 h indicative of interaction. Colony color was also scored(see Figs. 3B and 4B). Plasmids encoding interacting proteins SV40T antigen and murine P53 (AT and BP53) were cotransformed aspositive controls, and AT was cotransformed with BLam encod-ing non-interacting human lamin protein for negative controls.Autonomous �-galactosidase activation was tested for all con-structs but only detected for BToCV p27.

Using urea–SDS and tricarboxylic acid protein extraction meth-ods according to manufacturer’s protocols (Clontech Laboratories,Inc., Mountain View, CA), we were able to detect protein expressionfrom all of the capsid component fusions (AHsp, AP59, ACP, ACPm,and BHsp, BP59, BCP, and BCPm), but were unable to detect proteinexpression from P34 or P26 constructs (data not shown).

Interaction strength was quantified by measuring �-galactosidase activity and calculating �-galactosidase (�-Gal)units according to the manufacturer’s instructions (ClontechLaboratories, Inc., Mountain View, CA). Relative �-Gal units werecomputed by dividing the unit values by the average unit valueof the positive control set (AT + BP53) in the same experimentalreplicate. Representative data from one experiment with replicatesare shown in figures.

2.3. Sequence analyses and alignments

Crinivirus P26 sequences were compared and aligned using Vec-tor NTI AlignX software (Invitrogen Corp., Carlsbad, CA). Secondarystructures of P26 and P9 proteins were predicted using JPred 3software (Cole et al., 2008).

3. Results

3.1. Protein interaction matrix of six major LIYV-encodedproteins: P26 is a self-interacting protein

LIYV proteins P34, Hsp70h, P59, CP, CPm, and P26 were

assessed in all possible pairwise combinations (6 activation domainfusions x 6 binding domain fusions = 36 pairwise combinations) forinteractions in a yeast-two-hybrid system. Only the AP26 + BP26combination grew on medium- and high-stringency media selec-tive for protein interaction and exhibited �-galactosidase activity in

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lter lift assays, as did yeast transformed with the positive controlnteraction pair (AT + BP53).

.2. N- and C-terminal truncation series to map the P26 regionesponsible for self-interaction

Analyses of LIYV P26 amino acid (aa) sequences so far haveot revealed any domains or motifs predicting protein interaction.

n order to determine regions important for P26 self-interaction,e generated a series of N- and C-terminal truncations (Fig. 1A).

runcations fused to the GAL4 activation domain were tested fornteractions with the full-length LIYV P26 binding domain fusionBLIYV P26). N- and C-terminal portions of the LIYV P26 proteinN-half, aa 1-114; C-half, aa 115–227) and a series of N-terminalruncations (numbered A1–A8) and C-terminal truncations (num-ered A9–A16) were tested. Interaction strength was qualitativelyssessed and ranked by colony growth on low-, medium-, and high-

tringency media (L, M, and H) and by �-galactosidase activity.rowth on L, M, and H media and �-galactosidase activity in at least

hree replicated experiments was considered indicative of strongnteraction, growth on L and M and �-galactosidase activity indi-ated weaker interaction, growth on L media and weak growth on M

ig. 1. Mapping regions important for LIYV P26 self-interaction. (A) Interactionsf AD fusions to LIYV P26 C- and N-terminal truncations with full-length P26 BDusion. Interactions are indicated by color gradations from red (no interaction) toark blue (strong interaction). Strong interaction was scored by colony growth andn low-, medium-, and high-stringency selective media (LMH), while weak inter-ction indicates growth on low- and medium-stringency (LM), and possible weaknteraction indicated by occasional �-galactosidase activity detected on coloniesrown on low-stringency selection but which failed to grow under stronger selectionL). Pale colony color was observed for cotransformants with constructs A6 and A7,sually indicative of protein interaction, but no �- or �-galactosidase activity wasetected in these colonies. (B) Quantitative assessments of interaction strength wereerformed by measuring �-galactosidase activity in yeast culture supernatants. Bars

ndicate �-galactosidase activity relative to that of the control AT + BP53 interactingair (set to 1). Error bars show standard deviations. Numbers above bars indicateultures which grew in selective media (except for AT + BLam negative control pair,rown in nonselective media), over total attempted in this experiment.

rch 145 (2009) 293–299 295

media with some �-galactosidase activity indicated possible weakinteraction, and growth on only L media with no �-galactosidaseactivity detected indicated no protein interaction (Fig. 1A). Quan-titative assessments of �-galactosidase activity, measured as anindicator of interaction strength, paralleled qualitative observa-tions (Fig. 1B). In quantitative and qualitative assays, interactioncomparable to that of the full-length LIYV P26 was found only fortruncation A1 (aa 24–227). No interaction was detected for any ofthe other N-terminal truncations, although cotransformants withtruncations A6 (aa 161–227) and A7 (aa 184–227) consistentlyexhibited pale colony color, usually indicative of protein interac-tion. The C-terminal truncations including the N-half exhibitedweakened interaction with BLIYV P26, except for truncations A9(aa 1–23), and A10 (1–46), which did not retain detectable interac-tion properties. Cotransformants with truncation A15 (aa 1–183)grew poorly or not at all on selective media, indicating weak or nointeraction.

3.3. Self-interaction of P26 orthologs

The LIYV P26 amino acid sequence exhibits low similarity toother Crinivirus P26 sequences (Fig. 2), although alignment of sub-sets of P26 orthologs may result in detection of conserved aminoacid residues (Dolja et al., 2006). However, secondary structure pre-dictions of Crinivirus P26 proteins indicated similar folding patternsfor the orthologous proteins, but again the LIYV-encoded proteinappeared to be most divergent among the orthologs (Fig. 2B). Thepredicted secondary structure of LIYV P26 is most similar to thoseof crinivirus orthologs in the N-terminal regions (aa 24–71 and92–114), corresponding to regions which appeared to be importantfor LIYV P26 self-interaction. Previous biochemical fractionationdata indicated that BPYV and CYSDV P26 orthologs retain some bio-chemical properties similar to the LIYV protein (Stewart et al., 2009)so we tested whether orthologs had protein interaction propertiessimilar to LIYV P26.

P26 proteins encoded by orthologous genes in BPYV, CYSDV,LCV, and ToCV were tested in all pairwise combinations for interac-tions (Fig. 3A). The BToCV p27 fusion caused autonomous activationof reporter genes by single transformation and cotransformationwith any of the other plasmids, and consequently could not beassayed for interactions using this system. However, each of theother P26 orthologs exhibited strong self-interaction, and noneinteracted with orthologous proteins (Fig. 3A). Qualitative assess-ment of interactions indicated that each of the P26 orthologsexhibited strong self-interaction (determined by colony growthon L, M, and H media and detection of �-galactosidase activ-ity in these colonies; Fig. 3B). BPYV P26 cotransformant coloniesappeared up to 7 days later than LIYV P26 colonies on medium-and high-stringency selective plates and grew more slowly, butalways exhibited intense blue coloration in filter lift assays for�-galactosidase activity (data not shown). LCV and CYSDV P26cotransformants also grew more slowly than corresponding LIYVP26 colonies and always exhibited �-galactosidase activity, butwith less intense coloration than BPYV P26 (data not shown).Interaction strength was quantified by assaying �-galactosidaseactivity. LIYV, CYSDV, and LCV P26 orthologs exhibited similarself-interaction strengths, while BPYV P26 self-interaction mea-surements were over 4 times higher (Fig. 3C).

3.4. LIYV P5 and P9 protein interaction testing

Since we did not identify interactions of P26 with five otherlarge LIYV proteins, we further examined possible interactions withLIYV RNA2-encoded small proteins, P5 and P9. We tested theseproteins for interactions with each other and with the six LIYV pro-teins described above using mixtures of eight fusion constructs.

296 L.R. Stewart et al. / Virus Research 145 (2009) 293–299

Fig. 2. Comparisons of Crinivirus P26 orthologs. (A) P26 amino acid sequences used for Y2H assays aligned using Vector NTI AlignX (Invitrogen Corp., Carlsbad, CA). Residueshighlighted in yellow (L123) are conserved among all the P26 sequences (LIYV, BPYV, CYSDV, LCV, and ToCV); blue or green coloration indicates amino acid identity orsimilarity, respectively, across subsets of the aligned sequences. (B) Predicted secondary structures of Crinivirus P26 orthologs, computed using JPred3 (Cole et al., 2008).Residue positions predicted to participate in alpha helix formation are highlighted in red, while positions predicted to participate in beta sheet structures are highlighted inyellow. For each of the P26 orthologs, three lines showing secondary structure predictions represent the Jnet PSIBLAST pssm profile prediction (bottom), the Jnet hmm profileprediction (middle), and the final secondary structure prediction based on combining the predictions of the two programs (top). Amino acid sequences used for secondarysequence predictions correspond to GenBank sequences translated for analyses were: LIYV sequence U15441 (Klaassen et al., 1995), BnYDV sequence NC 01561 (Martine kberryA equens al., 20

PommNefoPwii

ttsCpcOaptc

t al., 2008), BPYV sequence AY330919 (Tzanetakis and Martin, 2004), BYVV (BlacY242078 (Aguilar et al., 2003), LCV sequence described herein (Table S2), PYVV sequence AY488138 (Tzanetakis et al., 2005), SPCSV sequence AJ428555 (Kreuze et

5 and P9 fusions were cotransformed with reciprocal mixturesf the eight protein fusion constructs (either Amix, containing aixture of AP34, AP5, AHsp, AP59, AP9, ACP, ACPm, and AP26 plas-ids; or Bmix containing a similar mixture of B-plasmids; Fig. 4A).o interactions were detected for AP5 or BP5 combinations. How-ver, interactions were detected for AP9 and BP9. We tested P9or self-interaction and for interaction with mixtures of all of thether LIYV proteins tested in this study except for P9 (mix-P9).9 + mix-P9 cotransformations exhibited no positive interactions,hile AP9 + BP9 cotransformants exhibited �-galactosidase activ-

ty and growth at all qualitative stringency levels indicating strongnteraction (Fig. 4B).

Quantitative assessment of �-galactosidase activity indicatedhat the P9 self-interaction strength was low (Fig. 4C) comparedo LIYV P26 self-interaction strength. Comparisons of publishedequences of viruses in the family Closteroviridae and in the genusrinivirus indicated that ORFs encoding an approximately 9 kDarotein are present in a similar position in all genomes of theriniviruses (between P59 and CP-encoding ORFs), but that no such

RF is predicted for viruses in other genera. Amino acid sequencelignment of the predicted P9 protein orthologs indicated that theredicted proteins contain conserved N-terminal, central, and C-erminal regions, divided by two internal regions which are notonserved across species in the genus (Fig. 5A). Secondary struc-

yellow vein virus) sequence AY776335 (Tzanetakis et al., 2005), CYSDV sequencece AJ508757 (Livieratos et al., 2004), SPaV (Strawberry pallidosis-associated virus)02), and ToCV sequence AY903448 (Wintermantel et al., 2005).

ture predictions also indicated similarities among the P9 orthologs,suggesting three conserved short alpha helices (Fig. 5B).

4. Discussion

Here we have studied protein interaction properties as a wayto help elucidate properties of crinivirus-encoded proteins. Apply-ing a Y2H system to assess protein interactions, we show that twocrinivirus-specific proteins, P26 and P9, show strong propensityto self-interact. Both P26- and P9-encoding ORFs are conservedamong members of the genus Crinivirus, but readily identifiableorthologs in other members of the family Closteroviridae are lack-ing. We performed further experiments to map the LIYV P26interaction domain to the N-terminal region and tested for conser-vation of protein interaction capacity in P26 orthologs. Even thoughprimary sequence similarity of the orthologs is very low, their pre-dicted secondary structures are more conserved. The BPYV, CYSDV,and LCV Crinivirus P26 proteins retained specific self-interaction

capacity.

Due to the lack of efficient reverse-genetics systems for studyof crinivirus genotypes in planta, little is currently known aboutthe functions either of P26 or P9 in crinivirus infections, but theirconservation among all members of the genus and self-interaction

L.R. Stewart et al. / Virus Resea

Fig. 3. Pairwise interactions of Crinivirus P26 orthologs. (A) Matrix of pairwisecombinations tested for protein interactions by yeast-two-hybrid assays. Pairs forwhich reporter upregulation was observed are highlighted in yellow. (B) AH109yeast cotransformants encoding LIYV protein fusions streaked on media with low-,medium-, and high-stringency selection. (C) Relative interaction strength of pro-tda

pucecdhso(tbf(

and some additional proteins (Hsp70h, L-Pro, p6, p64, CPm, CP,

ein combinations, determined as described for Fig. 1. Error bars indicate standardeviations. Numbers above bars indicate cultures which grew over total culturesttempted in this experiment.

roperties are intriguing. LIYV P26 has been shown to induce anique cytopathology, plasmalemma deposits (PLDs) that are asso-iated with virus particles in both plants and protoplasts (Stewartt al., 2009). We hypothesized that P26 might interact with virionomponents due to the association of PLDs with virus particles, butid not find evidence of such an interaction in the work describedere. However, the self-interaction observed for LIYV P26 is con-istent with the aggregation properties of LIYV P26 previouslybserved in localization and biochemical fractionation experimentsStewart et al., 2009). This result is also consistent with observa-

ions in other viruses where cytopathology-inducing proteins haveeen shown to have strong self-interaction properties, as is the caseor the Citrus tristeza virus amorphous inclusion body protein, p20Gowda et al., 2000).

rch 145 (2009) 293–299 297

Interestingly, although PLDs have not been found for infec-tions by other criniviruses studied so far, including BPYV, CYSDV,and ToCV (Medina et al., 2003), specific self-interaction propertiesappear to be conserved. Previously, we described that biochemi-cal fractionation patterns are also conserved among crinivirus P26orthologs despite substantial sequence divergence, although local-ization patterns similar to the LIYV P26 protein were not observed(Stewart et al., 2009). Whether the orthologs retain similar bio-logical functions remains to be determined. The importance of theN-terminal region of P26 for self-interaction corroborate previ-ously reported results in which GFP fusions to the N-terminus ofthe complete P26 protein exhibited partial localization to the cellperiphery with loss of aggregation properties, while GFP fusion tothe C-terminus of intact P26 retained aggregation properties but nolonger localized to the cell periphery (Stewart et al., 2009). Incom-plete localization of each GFP fusion suggested that GFP mightmask or interfere with properties of the P26 protein nearest to thefusion site, although self-interaction was retained when P26 wasfused at the N-terminus to smaller GAL4 halves for the Y2H assaysdescribed here. Interestingly, N-terminal regions shown here to bemost similar across the P26 orthologs in predicted secondary struc-ture correspond to regions important for LIYV P26 self-interaction.C-terminal regions between amino acids 161–183 have a verystrong interaction disruption effect in these experiments. Aminoacid residues 161–183 are not especially hydrophobic, and thecharge distribution is not strikingly different from that of surround-ing amino acids, so that we are currently unable to account for theeffect of these amino acids. Exposure of these residues may alterlocalization such that interaction is no longer detected in a yeast-two-hybrid system, or alternatively, may cause misfolding of theprotein such that protein interaction domains are no longer foldedin a manner conducive to this property.

Potential roles of P9 are unexplored at present, but this proteinis predicted for all sequenced member of the genus Crinivirus sofar, despite substantial variability in the number of small proteinspredicted to be encoded by each member of the genus. Whether P9proteins across the genus retain self-interaction properties remainsto be determined, but its conserved presence and structure suggeststhat it may perform some Crinivirus-specific role(s) in the infectioncycle and presents it as a candidate for future studies.

The yeast-two-hybrid system has limitations which mayaccount for the failure to detect some expected interactions, suchas interactions of the capsid proteins, in these experiments. Itis limited to addressing binary interactions, and it is possibletherefore that viral RNA or multiple components are necessary todetect interactions between the capsid protein components. Mul-tiple components or host factors might also be involved in thehypothesized interactions between LIYV P26 and capsid compo-nents. Additional limitations might arise from disruption of proteinproperties or interactions by the Y2H fusions, or other features ofthis Y2H system such as requirements for nuclear entry to upreg-ulate reporter gene expression. It is also possible that sequencesfrom the RNA criniviruses are misprocessed with the additionalnuclear phase or do not efficiently express translatable transcriptsfrom DNA templates. Reports of closterovirus protein interactionsin the literature support these possibilities, since some but not allexpected interactions have been detected. For CTV, CP–CP inter-actions were detected by Y2H assays, but no self-interactions orinteractions with CP were detected for the CPm protein (Gowda etal., 2000). BYV Hsp70h and p20 (which is associated with virions)were tested for interactions with each of the capsid components

p20, and p21—an RNA-binding protein, silencing suppressor, andhomolog of CTV p20) by Y2H. In these experiments, interactionsbetween Hsp70h and p20 and p20–p20 were detected, but no inter-actions between Hsp70h and other capsid components, p64, CP,

298 L.R. Stewart et al. / Virus Research 145 (2009) 293–299

Fig. 4. LIYV P5 and P9 yeast-two-hybrid interaction tests. (A) P5 and P9 fusions tested with mixtures of plasmids. Combinations highlighted in yellow were positive forinteraction at low, medium, or high selection stringencies as indicated. Bmix = equimolar mixture of BP34, BP5, BHsp, BP59, BP9, BCP, BCPm, and BP26 plasmids. Amix = similarc equimD teracm igh-sta LIYV Pa cate c

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ombination with activation domain fusion plasmids. Bmix-P9 and Amix-P9 wereata represent results from at least three experimental replicates, with positive inedia. (B) Colony growth of the interacting LIYV P9 pair on low-, medium-, and h

nd negative control non-interacting pair AT + BLam. (C) Interaction strength of thectivity set to 1. Error bars represent standard deviations. Numbers above bars indi

r CPm were detected (Prokhnevsky et al., 2002). Our attempts tose bimolecular fluorescence complementation (BiFC) to confirm

nteraction properties detected by yeast-two-hybrid in plant cellsere uninformative. All combinations with BPYV P26 and ToCV

ig. 5. Comparisons of Crinivirus P9 ortholog amino acid sequences and predicted secondlosum 62mt matrix in Vector NTI AlignX (Invitrogen Corp., Carlsbad, CA). Identical residre highlighted in green. (B) Secondary structure predictions of Crinivirus P9 orthologs usnd predictions were from GenBank accessions: LIYV NC 003618 (Klaassen et al., 1995),ere; BnYDV NC 010561 (Martin et al., 2008), BPYV NC 005210 (Tzanetakis and Martin, 2003), PYVV NC 006063 (Livieratos et al., 2004), SPaV NC 005896 (Tzanetakis et al., 2005nd ToCV AY903448 (Wintermantel et al., 2005).

olar mixtures of all the aforementioned plasmids except BP9 or AP9, respectively.tions detected by �-galactosidase filter lift assays and colony growth on selectiveringency selective media, compared to positive control interacting pair AT + BP539 pair relative to the control protein pairs, with AT + BP53 average �-galactosidase

ultures which grew over total attempted in the experiment represented.

P27 proteins fused to the N-terminus of yellow fluorescent pro-tein (YFP) resulted in strong YFP detection, while all combinationswith CYSDV P26, LCV P26, LIYV P26, or LIYV P9 resulted in low orno YFP detection. Since BPYV and ToCV proteins accumulated at

ary structures. (A) Amino acid alignment of Crinivirus P9 proteins generated using aues at each position are highlighted in blue, while similar residues at each positioning JPred 3 (Cole et al., 2008), as described for Fig. 2. Sequences used for alignmentP9 sequence identical to pSP6 clone P9 sequence used for experiments described004), BYVV NC 006963 (Tzanetakis et al., 2006), CYSDV NC 004810 (Aguilar et al.,

), SPCSV AJ428555 (Kreuze et al., 2002), TICV EU625351 (Jacquemond et al., 2009),

Resea

mpeCptiatfgpp

A

NnagC

tDRNDsP

A

t

R

A

A

C

C

C

D

L.R. Stewart et al. / Virus

uch higher levels than the other P26 orthologs or the LIYV P9roteins, the results appeared to reflect protein accumulation lev-ls (in a directional manner, since fusions of these proteins with the-terminus of YFP did not have a similar effect) rather than bona fiderotein interaction properties (data not shown). However, despitehe limitations of the yeast-two-hybrid system, it has been effectiven elucidating crinivirus protein interaction properties. As methodsre developed to assess the functions of crinivirus proteins in planta,he interaction properties of P9 and P26 proteins can be testedor functional relevance in the context of infection using reverse-enetics approaches. These studies utilize current technologies torovide novel insight into two self-interacting Crinivirus-specificroteins, P26 and P9.

cknowledgements

This work was funded primarily by a grant from the USDARICGP. It was also funded in part by a University of Califor-ia, Davis Jastro-Shields student research grant to L. R. Stewartnd a Korea Research Foundation Grant funded by the Koreanovernment (MOEHRD, Basic Research Promotion Fund KRF-2006-00075) to M. S. Hwang.

Many thanks to Haley Dequine and Gabriel Craig for their assis-ance in carrying out the Y2H screens and quantitative assays, tors. James Ng and Nida Salem (UC Riverside) for providing LCV viralNA and P26 sequence information prior to publication, to Dr. Jesusavas-Castillo (EELM, Spain) for providing the ToCV P27 clone, tor. Kentaro Inoue (UC Davis) for assistance in assessing secondary

tructures, and to Tyler Chandler for cloning the BPYV and CYSDV26 sequences used in this project.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.virusres.2009.07.021.

eferences

granovsky, A.A., Lesemann, D.E., Maiss, E., Hull, R., Atabekov, J.G., 1995. “Rat-tlesnake” structure of a filamentous plant RNA virus built of two capsid proteins.Proc. Natl. Acad. Sci. U.S.A. 92 (7), 2470–2473.

guilar, J.M., Franco, M., Marco, C.F., Berdiales, B., Rodriguez-Cerezo, E., Truniger, V.,Aranda, M.A., 2003. Further variability within the genus Crinivirus, as revealedby determination of the complete RNA genome sequence of Cucurbit yellowstunting disorder virus. J. Gen. Virol. 84 (Pt 9), 2555–2564.

ole, C., Barber, J.D., Barton, G.J., 2008. The Jpred 3 secondary structure predictionserver. Nucleic Acids Res. 36 (Web Server issue), W197–W201.

osta, A.S., 1976. Whitefly-transmitted plant diseases. Annu. Rev. Phytopathol. 14,429–449.

uellar, W.J., Tairo, F., Kreuze, J.F., Valkonen, J.P., 2008. Analysis of gene content insweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNAsilencing suppressor in only a few isolates: implications for viral evolution andsynergism. J. Gen. Virol. 89 (Pt 2), 573–582.

olja, V.V., Kreuze, J.F., Valkonen, J.P., 2006. Comparative and functional genomicsof closteroviruses. Virus Res. 117 (1), 38–51.

rch 145 (2009) 293–299 299

Gowda, S., Satyanarayana, T., Davis, C.L., Navas-Castillo, J., Albiach-Marti, M.R.,Mawassi, M., Valkov, N., Bar-Joseph, M., Moreno, P., Dawson, W.O., 2000. Thep20 gene product of Citrus tristeza virus accumulates in the amorphous inclusionbodies. Virology 274 (2), 246–254.

Jacquemond, M., Verdin, E., Dalmon, A., Guilbaud, L., Gognalons, P., 2009. Serolog-ical and molecular detection of Tomato chlorosis virus and tomato infectiouschlorosis virus in tomato. Plant Pathol. 58 (2), 210–220.

Klaassen, V.A., Boeshore, M.L., Koonin, E.V., Tian, T., Falk, B.W., 1995. Genomestructure and phylogenetic analysis of lettuce infectious yellows virus, awhitefly-transmitted, bipartite closterovirus. Virology 208 (1), 99–110.

Kreuze, J.F., Savenkov, E.I., Valkonen, J.P., 2002. Complete genome sequence andanalyses of the subgenomic RNAs of sweet potato chlorotic stunt virus revealseveral new features for the genus Crinivirus. J. Virol. 76 (18), 9260–9270.

Livieratos, I.C., Eliasco, E., Muller, G., Olsthoorn, R.C., Salazar, L.F., Pleij, C.W., Coutts,R.H., 2004. Analysis of the RNA of Potato yellow vein virus: evidence for a tripar-tite genome and conserved 3′-terminal structures among members of the genusCrinivirus. J. Gen. Virol. 85 (Pt 7), 2065–2075.

Martin, G., Velasco, L., Segundo, E., Cuadrado, I.M., Janssen, D., 2008. The completenucleotide sequence and genome organization of bean yellow disorder virus, anew member of the genus Crinivirus. Arch. Virol. 153 (5), 999–1001.

Medina, V., Rodrigo, G., Tian, T., Juarez, M., Dolja, V.V., Achon, M.A., Falk, B.W., 2003.Comparative cytopathology of crinivirus infections in different plant hosts. Ann.Appl. Biol. 143, 99–110.

Medina, V., Sudarshana, M.R., Tian, T., Ralston, K.S., Yeh, H.H., Falk, B.W., 2005. TheLettuce infectious yellows virus (LIYV)-encoded P26 is associated with plas-malemma deposits within LIYV-infected cells. Virology 333 (2), 367–373.

Ng, J.C., Falk, B.W., 2006. Bemisia tabaci transmission of specific Lettuce infectiousyellows virus genotypes derived from in vitro synthesized transcript-inoculatedprotoplasts. Virology 352 (1), 209–215.

Pinto, R.L., Hoeffert, L.L., Fail, G.L., 1988. Plasmalemma deposits in tissues infectedwith Lettuce infectious yellows virus. J. Ultrastruct. Mol. Struct. Res. 100 (3),245–254.

Polston, J.E., Anderson, P.K., 1997. The emergence of whitefly-transmitted gemi-niviruses in tomato in the western hemisphere. Plant Dis. 81, 1358–1369.

Prokhnevsky, A.I., Peremyslov, V.V., Napuli, A.J., Dolja, V.V., 2002. Interactionbetween long-distance transport factor and Hsp70-related movement proteinof Beet yellows virus. J. Virol. 76 (21), 11003–11011.

Satyanarayana, T., Gowda, S., Ayllon, M.A., Dawson, W.O., 2004. Closterovirus bipo-lar virion: evidence for initiation of assembly by minor coat protein and itsrestriction to the genomic RNA 5′ region. Proc. Natl. Acad. Sci. U.S.A. 101 (3),799–804.

Stewart, L.R., Medina, V., Sudarshana, M.R., Falk, B.W., 2009. Lettuce infectious yellowsvirus-encoded P26 induces plasmalemma deposit cytopathology. Virology 388(1), 212–220.

Tian, T., Rubio, L., Yeh, H.H., Crawford, B., Falk, B.W., 1999. Lettuce infectious yel-lows virus: in vitro acquisition analysis using partially purified virions and thewhitefly Bemisia tabaci. J. Gen. Virol. 80 (Pt 5), 1111–1117.

Tzanetakis, I.E., Martin, R.R., 2004. Complete nucleotide sequence of a strawberryisolate of Beet pseudoyellows virus. Virus Genes 28 (3), 239–246.

Tzanetakis, I.E., Reed, J., Martin, R.R., 2005. Nucleotide sequence, genome organiza-tion and phylogenetic analysis of Strawberry pallidosis associated virus, a newmember of the genus Crinivirus. Arch. Virol. 150 (2), 273–286.

Tzanetakis, I.E., Susaimuthu, J., Gergerich, R.C., Martin, R.R., 2006. Nucleotidesequence of Blackberry yellow vein associated virus, a novel member of theClosteroviridae. Virus Res. 116 (1–2), 196–200.

Wintermantel, W.M., 2004. Emergence of greenhouse whitefly (Trialeu-rodes vaporariorum) transmitted criniviruses as threats to vegetableand fruit production in North America. Am. Phytopathol. Soc. (June),www.apsnet.org/online/feature/whitefly/.

Wintermantel, W.M., Wisler, G.C., Anchieta, A.G., Liu, H.Y., Karasev, A.V., Tzane-takis, I.E., 2005. The complete nucleotide sequence and genome organization

of tomato chlorosis virus. Arch. Virol. 150 (11), 2287–2298.

Wisler, G.C., Duffus, J.E., Liu, H.Y., Li, R.H., 1998. Ecology and epidemiology ofwhitefly-transmitted closteroviruses. Plant Dis. 82, 270–276.

Yeh, H.H., Tian, T., Rubio, L., Crawford, B., Falk, B.W., 2000. Asynchronous accumu-lation of lettuce infectious yellows virus RNAs 1 and 2 and identification of anRNA 1 trans enhancer of RNA 2 accumulation. J. Virol. 74 (13), 5762–5768.