Báo cáo khoa học: Interaction of Sesbania mosaic virus movement protein with the coat protein – implications for viral spread Soumya Roy Chowdhury and Handanahal Subbarao Savithri docx

In the first type, MPs interact with viral RNA to form a movement complex M complex, which is transported from cell to cell, as in tobacco mosaic Keywords coat protein; protein–protein in

with the coat protein – implications for viral spread

Soumya Roy Chowdhury and Handanahal Subbarao Savithri

Department of Biochemistry, Indian Institute of Science, Bangalore, India

Introduction

Plants have an elaborate communication system that

permits transport of macromolecules from one cell to

another Plant viruses have evolved mechanisms to

manipulate the same resident communication system

and redirect it in such a way that the viral genome is

transported from one cell to another, leading to spread

of infection The virus-encoded movement protein

(MP), in association with other viral and host factors

called ancillary proteins, plays a central role in this

process The MP–genome complex, or, in some cases,

assembled virus particles, interacts with the compo-nents of plasmodesmata and dilates the openings to permit passage through the cell wall [1,2] Although MPs are not conserved across genera, they perform similar functions [3] In terms of the nature of the nucleoprotein complex that moves from cell to cell, plant viruses may be broadly divided into two types [4] In the first type, MPs interact with viral RNA to form a movement complex (M complex), which is transported from cell to cell, as in tobacco mosaic

Keywords

coat protein; protein–protein interaction;

recombinant movement protein;

sobemovirus

Correspondence

H S Savithri, Department of Biochemistry,

Indian Institute of Science, Bangalore

560012, India

Fax: +91 8023600814

Tel: +91 8022932310

E-mail: bchss@biochem.iisc.ernet.in

(Received 10 June 2010, revised 28

September 2010, accepted 1 November

2010)

doi:10.1111/j.1742-4658.2010.07943.x

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of South-ern bean mosaic virus within the Sobemovirus genus With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent man-ner Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N-terminal 49 amino acid segment Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require

MP and NV⁄ coat protein for cell-to-cell movement

Structured digital abstract

l MINT-8050243 : p53 (uniprotkb: P02340 ) physically interacts ( MI:0915 ) with T-Ag (uni-protkb: P03070 ) by two hybrid ( MI:0018 )

l MINT-8050226 : MP (uniprotkb: Q9EB09 ) physically interacts ( MI:0915 ) with CP (uni-protkb: Q9EB06 ) by two hybrid ( MI:0018 )

Abbreviations

CP, coat protein; CPMV, cowpea mosaic virus; GnHCl, guanidine hydrochloride; GST–MP, recombinant MP expressed in E coli with an N-terminal glutathione sulfur transferase tag; NV, native virus; M complex, movement complex formed by MP with viral genomic RNA;

MP, movement protein; rMP, recombinant MP expressed in E coli with an N-terminal histidine tag; SBMV, Southern bean mosaic virus; SeMV, Sesbania mosaic virus; TMV, tobacco mosaic virus; Y2H, yeast two-hybrid.

virus (TMV) TMV has been shown to be transported

as a replication complex that contains MP, viral

repli-case and genomic RNA [5] In the second type, intact

virus particles are transported through MP-containing

tubules, as observed in cowpea mosaic virus (CPMV)

[6] However, MPs that are known to form an M

com-plex can also form tubules [7], and MPs that form

tubules can also bind to RNA [8,9]

An extensive analysis of the function of MPs of

viruses from various genera has shown that

taxonomi-cally different viruses may use the same strategy, while

closely related viruses may use different strategies, and

some may use more than one strategy for the spread

of infection [3] It is also possible that viruses may use

different strategies depending on the host they infect

[10] The mechanism of virus movement is therefore

diverse and complex, involving several factors [11]

Sobemoviruses are plant RNA viruses that are

named after their type species, Southern bean mosaic

virus (SBMV) Viruses belonging to this genus are

ico-sahedral particles of approximately 30 nm in diameter

The viral capsid is made up of 180 identical coat

pro-tein (CP) subunits organized with T = 3 icosahedral

symmetry, and Encapsidates a single molecule of

geno-mic RNA The genogeno-mic RNA is a single-stranded

mes-senger-sense molecule, approximately 4–4.5 kb in size

The 5¢ terminus of the RNA has a genome-linked

pro-tein (VPg), and the 3¢ end lacks a poly(A) tail

Sobem-oviruses infect plants from 15 families, including

dicotyledonous and monocotyledonous species [12]

The first sobemovirus, SBMV, was reported from

Lou-isiana and California, USA, in 1943 [13] Later it was

reported that sobemoviruses occur all over the world,

infecting plants in countries from Scandinavia to New

Zealand and throughout tropical Africa, North

Amer-ica and South East Asia In susceptible hosts,

sobem-oviruses cause severe diseases with recurrent economic

losses For example, rice yellow mottle virus is

respon-sible for the most rapidly spreading disease of rice in

Africa [12] Previous studies on sobemoviruses have

shown that the protein coded by ORF1 of rice yellow

mottle virus [14] and Southern cowpea mosaic virus

[15] is essential for cell-to-cell movement More

recently, it was reported that the ORF1-encoded

pro-tein of Cocksfoot mottle virus (CfMV), designated P1,

is essential for systemic spread of the virus [16]

Fur-ther, the ORF1-encoded product of rice yellow mottle

virus has been implicated as a RNA silencing

suppres-sor [17,18] These results suggest that ORF1-encoded

proteins function as MPs in sobemoviruses However,

the molecular mechanism of cell-to-cell movement in

sobemoviruses has not been investigated Other

virus-encoded ancillary proteins that may interact with

sobemoviral MPs and assist in cell-to-cell or systemic movement of the virus have not yet been identified It

is not known whether sobemoviruses use TMV-type or CPMV-type movement strategies Functional charac-terization of sobemoviral MPs and understanding of the role of ancillary proteins⁄ domains in cell-to-cell movement may assist in identification of genome seg-ments that could be targeted for developing antiviral strategies for this particular virus group

Sesbania mosaic virus (SeMV) belongs to the Sob-emovirus genus, and was first identified from infected Sesbania grandiflora pers agathi on farms around Tirupati, Andhra Pradesh, India The 3D structure of the purified virus has been determined, and it was shown to be an icosahedral virus with a diameter of

30 nm comprising 180 identical CP subunits [19,20] The SeMV genome is 4149 nucleotides long, and encodes four potential overlapping ORFs [21] Com-parison of the nucleotide and the deduced amino acid sequences of SeMV ORFs with those of other sobem-oviruses revealed that SeMV is closest to the South-ern bean mosaic virus Arkansas isolate (SBMV-Ark) [21] The mechanisms of SeMV assembly and poly-protein processing have been reported previously [22–25]

In the present study, the ORF1 gene encoding the SeMV MP was cloned and over-expressed in Escheri-chia coliin fusion with a hexahistidine or a glutathione sulfur transferase (GST) tag The recombinant proteins were shown to interact with native virus (NV) using pull-down assays and ELISA Further, studies on dele-tion mutants of MP were performed to determine the domain responsible for the interaction of MP with CP using ELISA and yeast two-hybrid (Y2H) assays Deletion of the N-terminal 49 amino acids of the SeMV MP drastically reduced the interaction between the two proteins To our knowledge, this is the first report demonstrating the interaction between MP and

CP of a sobemovirus, suggesting that SeMV might belong to the class of viruses that require both MP and CP for cell-to-cell movement

Results

In silico analysis of MPs

In order to examine the similarity among the MPs of sobemoviruses and other plant viruses, in silico analysis was performed The gene sequences were obtained from the National Center for Biotechnology Information and GenBank, and multiple sequence alignment was per-formed using Clustal W2 (http://www.ebi.ac.uk/Tools/ clustalw2/index.html) The results obtained are shown

in Table 1 The SeMV MP showed no significant

sequence similarity with MPs of viruses from other

gen-era Within sobemoviruses, the sequence of the SeMV

MP was closest to that of the SBMV-Ark MP (32%

sequence identity), and the identity with MPs of other

sobemoviruses was not significant The secondary

struc-ture of SeMV MP was predicted using the

PredictPro-tein server (http://www.predictproPredictPro-tein.org/) [26] As

shown inFig 1, the SeMV MP was predicted to be a

primarily a-helical protein The predicted percentages

of a helix, b sheet and coil were 49%, 25% and 26%,

respectively The potential involvement of

post-transla-tional modification of viral MPs in regulation of their

transport mechanism was first suggested in view of

finding that the 30 kDa MP of TMV is phosphorylated

within host cells Other viral MPs, such as those of

tomato mosaic virus and potato leafroll virus, were

sub-sequently also shown to be phosphorylated during the

infection process [27,28] One consequence of the

phos-phorylation event on MP is that it could result in the

unloading of the viral genome from the M complex

after it enters the neighbouring cell through

plasmodes-mata [29–31] However, other reasons for

phosphoryla-tion of MPs could also exist that have yet to be

identified Nevertheless, the fact remains that MPs are

sometimes post-translationally modified by

phosphory-lation Therefore, a search for the presence of potential

phosphorylation sites in the SeMV MP was performed

using the netphos 2.0 server (http://www.cbs.dtu.dk/

services/NetPhos/) RNA binding sites and other motifs

were searched using the Block search program (http://

blocks.fhcrc.org/blocks/blocks_search.html) The results suggested the presence of a nucleic acid binding domain

in the C-terminal segment of the SeMV MP (Fig 1, grey box) and a high density of predicted phosphoryla-tion sites at the C-terminus of the protein (Fig 1, yellow box) However, no conserved motif was found when the SeMV MP sequence was compared with other well characterized MPs

Over-expression of the SeMV MP in E coli and purification under denaturing conditions The SeMV MP gene was amplified and cloned into the pRSET C vector (Invitrogen, Carlsbad, CA, USA) and over-expressed in E coli as described in Experimental procedures The recombinant histidine-tagged MP was designated rMP The 20 kDa rMP, although expressed

in at a high level (Fig 2A), was mostly present in the insoluble fraction (Fig 2B) The rMP was therefore purified from the insoluble fraction under denaturing conditions using 6 m guanidine hydrochloride (GnHCl), and refolded by stepwise dialysis as described in Experi-mental procedures The purity of the protein was deter-mined by 12% SDS⁄ PAGE (Fig 2C) The refolded rMP was soluble and was used for further characterization

Table 1 Sequence comparison of SeMV MP with other MPs from

various genera.

Genus Virus species

Percentage identity with SeMV

Percentage similarity with SeMV Sobemovirus Sesbania mosaic virus 100 100

Southern bean mosaic

virus

Southern cowpea

mosaic virus

Lucerne transient streak

virus

Rice yellow mottle virus 9.1 12.3

Cocksfoot mottle virus 8 15.1

Tobamovirus Tobacco mosaic virus 11 19.9

Alfamovirus Cowpea chlorotic mottle

virus

Cucumovirus Cucumber mosaic virus 9.8 17.6

Fig 1 Prediction of the secondary structure of the SeMV MP S, sequence; P, secondary structure predicted using the PredictPro-tein server (http://www.predictproPredictPro-tein.org/) The grey boxes repre-sents the RNA binding motif The red boxes indicate cysteine residues and the yellow box indicates a high-propensity phos-phorylation site predicted by the NetPhos 2.0 server (http://www cbs.dtu.dk/services/NetPhos/).

Circular dichroism (CD) spectroscopy

The far-UV CD spectrum of the purified and refolded

rMP showed minima at 210 and 222 nm, suggesting

that the protein was folded and adopted a largely

a-helical conformation (Fig 3A) Analysis of the CD

spectrum using K2D2 software (http://www.ogic.ca/

projects/k2d2/) showed that rMP comprises more than

84% a-helical structure, compared to the predicted

helix content of 49% The thermal stability of the rMP

was monitored by measuring the molar ellipticity at

210 nm as a function of temperature The rMP had a

Tmof 65C (Fig 3A, inset)

Fluorescence spectroscopic analysis

The intrinsic fluorescence spectrum of rMP showed

maximum emission at 345 nm upon excitation at

280 nm, typical of a folded protein (Fig 3B) The

emission maximum showed a red shift to 365 nm upon

addition of 8 m urea due to exposure of the aromatic

residues to the solvent as the protein unfolded [30]

Another broad peak between 305 and 315 nm was also

observed upon urea denaturation (Fig 3B) Generally,

the fluorescence emission induced in proteins by

280 nm excitation is dominated by tryptophan

fluores-cence and the tyrosine emission is nearly undetectable

The tyrosine emission (305–315 nm) is observed only

when the protein is in the denatured state [32]

MP–NV interaction

NV or the CP is an important ancillary protein for the movement of many viruses within the host To deter-mine whether SeMV MP and NV interact with each other, pull-down assays were performed as described

in Experimental procedures A distinct band corre-sponding to CP was seen together with rMP in the eluted fraction (data not shown)

To confirm the interaction of MP with NV, a modi-fied ELISA was performed as described in Experimen-tal procedures ELISA plates were coated with NV, blocked, and rMP was added The interaction between the two proteins was monitored by using antibodies against rMP (Fig 4A) In a reverse experiment, rMP was immobilized on ELISA platea and NV was used

as the probe protein (Fig 4B) In both the experi-ments, BSA was used as a control Cross-reaction of the primary antibody to the immobilized protein was also tested by ELISA in the absence of the interacting proteins rMP interacted with NV in both the experi-ments (Fig 4A,B)

Nature of the MP–NV interaction

To determine the concentration dependence and nature

of the interaction between MP and NV, the same ELISA-based approach was used ELISA plates were coated with NV (5 lg), and, after blocking, increasing

Fig 2 Expression, solubility analysis and purification of rMP (A) The pRSET C-MP clone was transformed into E coli BL21 (DE3) cells The total lysate after isopropyl-b- D -thiogalactopyranoside induction was analysed by 12% SDS ⁄ PAGE Lanes U and I correspond to uninduced and induced total lysate, respectively The arrow indicates the position of the rMP band in the induced sample (lane I) (B) SDS ⁄ PAGE (12%)

of soluble (S) and pellet (P) fractions of rMP-expressing cells The arrow indicates the position of the rMP band (C) SDS ⁄ PAGE (12%) show-ing rMP purified by Ni-NTA affinity chromatography under denaturshow-ing conditions usshow-ing 6 M GnHCl (lanes 1 and 2) The arrow indicates the position of purified protein Lane M, protein molecular mass markers The gels were stained with Coomassie brilliant blue R250.

concentrations of rMP were added, and the ELISA

was performed The absorbance at 450 nm was plotted

as a function of rMP concentration (Fig 5A) The

results show that the interaction between NV and rMP

is concentration-dependent

Next, we wished to investigate the effect of pH on

MP–NV interaction Previously, it was shown that

SeMV particles are stable over a pH range of 3–10.4

[33] Similar studies with rMP showed that the protein was stable over a broad pH range and precipitated at

pH values > 9 [34] Therefore, to determine the opti-mal pH for interaction between the two proteins, rMP was dissolved in buffers at various pH values (Fig 5B), and incubated with NV-bound plates for 1 h Then the unbound rMP was removed and an ELISA was per-formed The reactions were performed in triplicate After the reaction, absorbance values obtained at

450 nm were plotted as a function of the pH of the

A

B

Fig 3 Biophysical characterization of refolded rMP (A) Far-UV CD

spectrum of rMP The molar ellipticity of rMP (0.5 mgÆmL)1) was

recorded from 190 to 300 nm in a 0.2 cm path-length cuvette with

a band width of 1 nm and response time of 1 s The thermal

stabil-ity of rMP was monitored by measuring the molar ellipticstabil-ity at

210 nm at various temperatures (inset) (B) Intrinsic fluorescence

spectra of rMP The intrinsic fluorescence spectrum of rMP was

measured by exciting the sample at 280 nm and observing the

fluo-rescence emission from 300 to 400 nm in the absence (solid line)

and presence of 8 M urea (dotted line) The emission maximum

showed a red shift to 365 nm upon addition of 8 M urea, and a

broad peak between 305 and 315 nm was also observed due to

emission by tyrosine residues that are exposed in the protein in the

denatured state.

A

B

Fig 4 Interaction of rMP with NV (A) ELISA of rMP and NV inter-action ELISA plates coated with NV (5 lg) (P1) were blocked with 10% skimmed milk in 1% NaCl ⁄ Pi (block) followed by addition of

5 lg of rMP (P2) The ELISA was performed using an anti-MP poly-clonal antibody (pAb to P2) and developed using anti-rabbit IgG con-jugated to horseradish peroxidise and DMB H 2 O 2 (sAb + Sub) The steps involved and the controls used are indicated on the figure BSA was used as a negative control (B) Reverse experiment in which ELISA plates were coated with rMP (P1) and probed with

NV (P2) Primary polyclonal antibodies against NV (pAb to P2) were used in this reaction, with controls similar to those used in (A).

buffer in which rMP was dissolved The optimal pH for interaction between the two proteins was between

pH 6.5 and 7.5, with a sharp decrease in both the acidic and alkaline ranges These observations suggest that the interaction between the two proteins is opti-mal near physiological pH

To monitor the effect of NaCl on the interaction of the two proteins, ELISA was performed as before, except that the rMP was dissolved with various concen-trations of NaCl and added to NV-bound ELISA plates Incubation of the SeMV MP or the NV with 1 m NaCl did not affect solubility or stability The absorbance at

450 nm was plotted as a function of NaCl concentration (Fig 5C) An increase in NaCl concentration did not affect the interaction of NV with rMP, suggesting that the interaction between the two proteins is strong

Expression of GST–MP The experiments described so far were performed using refolded rMP The MP-encoding gene was also cloned and over-expressed with an N-terminal GST tag as described in Experimental procedures The GST–MP fusion protein expressed in E coli BL21 (DE3) was sol-uble and of the expected size (45.4 kDa) The protein was purified using glutathione affinity chromatography, and was found to be homogeneous (Fig 6A, lanes E)

Protein–protein interaction between NV and GST–MP

To determine whether the soluble GST–MP interacts with NV in a manner similar to that of refolded rMP,

A

B

C

Fig 5 Analysis of the biochemical nature of the interaction between rMP and NV (A) Effect of rMP concentration on the NV–MP interac-tion NV-coated ELISA plates were incubated with increasing concen-trations of rMP after a blocking step The absorbance values obtained at 450 nm by ELISA with anti-MP polyclonal antibody were plotted as a function of rMP concentration (B) Effect of pH on the NV–MP interaction ELISA plates coated with NV (P1) were incu-bated with rMP (P2) in 50 m M buffers at various pH as indicated in the figure After incubation, the wells were washed, ELISA was performed using anti-MP polyclonal antibody (pAb to P2), and absorbance values at 450 nm were plotted as a function of pH rMP

in NaCl ⁄ Pi (pH 7.4) was used as a positive control The other controls used are indicated in the figure (C) Effect of NaCl on the NV–MP interaction ELISA plates coated with NV (P1) were blocked with 10% skimmed milk in PBS and incubated with rMP (P2) dissolved in

50 m M Tris ⁄ HCl buffer (pH 7.4) containing various concentrations of NaCl as shown in the figure and incubated for 1 h After extensive washing of the wells, ELISA was performed as described in Experimental procedures using anti-MP polyclonal antibody (pAb to P2) The absorbance values obtained at 450 nm were plotted as a function of NaCl concentration.

ELISA and GST pull-down experiments were

per-formed as described previously GST–MP was found

to interact with NV both in ELISA (Fig 6B) and

pull-down assays (data not shown) To rule out the

possi-bility that the interaction between GST–MP and NV is

due to interaction between GST and NV, a GST

blocking step was introduced in the ELISA-based

interaction assay (Fig 6B, last two columns) There

was no significant difference in the binding of GST–

MP to NV in the presence or the absence of GST, sug-gesting that the interaction is indeed between MP and

NV and not between GST and NV

Generation of deletion mutants of GST–MP The results presented so far clearly demonstrated that

MP interacts with NV It was of interest to map the region of MP that is responsible for this interaction For this purpose, a number of deletion mutant clones were constructed As described earlier, SeMV MP is primarily an a-helical protein The three predicted N-terminal three helices (Fig 1) were systematically deleted to generate ND16, ND35 and ND49 deletion mutant clones In addition, three C-terminal deletion mutant clones were also constructed: CD3, in which

a high-propensity phosphorylation site was removed, CD19 in which the predicted RNA binding domain was removed, and CD38, in which three cysteines and the nucleic acid binding domain were deleted (Fig 1)

The GST–MP deletion mutants were over-expressed

in E coli BL21(DE3)pLysS as described for the full length GST–MP All the mutant proteins were soluble and of the expected size The proteins were purified using glutathione affinity chromatography (Fig 7A)

Mapping of the SeMV MP domain necessary for interaction with NV

To determine which domains are involved in the inter-action of MP with NV, ELISA was performed as described previously ELISA plates were coated with

NV (5 lg) and blocked with 10% milk in NaCl⁄ Pi, followed by addition of various mutants as probe pro-teins The ELISA was performed using anti-rMP as the primary antibody In parallel, subsequent wells were incubated with GST and probed using polyclonal antibodies against GST to rule out the possibility of GST–NV interaction Determination of the absor-bance at 450 nm clearly showed that the N-terminal deletions have a pronounced effect on MP–NV inter-action Successive deletion of one, two and three pre-dicted helices from the N-terminus of MP reduced the interaction with NV by 51.5%, 66.4% and 80.1%, respectively, compared with the interaction between GST–MP and NV However, the interaction was not affected when C-terminal amino acids were deleted It may therefore be concluded that MP interacts with

NV via the N-terminal domains Similar observations were also made in pull-down experiments (data not shown)

A

B

Fig 6 Purification of GST–MP, and determination of the

interac-tion between GST–MP and NV by ELISA (A) Coomassie brilliant

blue-stained 12% SDS ⁄ PAGE gel showing GST–MP and GST

puri-fied by glutathione affinity chromatography Lanes are marked as

unbound protein (U), washed samples (W), eluted GST–MP (E) and

purified GST (G) Lane M, protein molecular mass markers Arrows

indicate the position of purified proteins (B) Protein–protein

interac-tion between NV and GST–MP An ELISA to measure the protein

interaction between NV (P1) and GST–MP (P2) was performed as

described in the legend to Fig 4B using anti-MP polyclonal antibody

(pAb to P2) Experimental steps and controls are indicated in the

figure An additional GST blocking step (GST) was included to rule

out the possibility of GST–NV interaction.

Y2H assays

All the results obtained so far were from in vitro

exper-iments To confirm these results, a Y2H assay was

performed using the Matchmaker system pGAD T7 (CP) and pGBK T7 (MP and deletion mutants) clones were obtained as described in Experimental procedures The pGAD T7 and pGBK T7 clones were transformed into the Saccharomyces cerevisiae AH109 strain in pairs

as indicated in Fig 7 pGBKT7-P53 (murine p53 fused

to GAL4 DNA BD) and pGADT7-T Ag (the SV40 large T-antigen fused to GAL4 DNA AD) that have been previously reported to interact in a Y2H assay [35] were used as a positive control in these experiments After transformation, growth was monitored on syn-thetic drop-out (SD) –Leu⁄ –Trp plates to confirm that both the plasmids were transformed into AH109 cells Subsequently, the transformed colonies were replica-plated on –Leu⁄ –Trp ⁄ –His (medium stringency), –Leu ⁄ –Trp⁄ –His ⁄ –Ade (high stringency), –Leu ⁄ –Trp ⁄ –His ⁄ + a-X-Gal (medium stringency with a-galactosidase) and –Leu⁄ –Trp ⁄ –His ⁄ –Ade ⁄ +a-X-Gal (high stringency with a-galactosidase) SD plates to determine the quality and strength of interaction between the SeMV MP or the deletion mutants and CP (Fig 8A,B)

AH109 cells co-transformed with pGBK T7 MP and pGAD T7 CP grew on all nutritional selection medium

up to the final level of selection (–Leu⁄ –Trp ⁄ –His ⁄ –Ade⁄ +a-X-Gal), similar to the positive control com-prising p53 and T-Ag (first two rows from the top in Fig 8A,B), suggesting that MP and CP also interact with each other under the ex vivo conditions of Y2H system However, the AH109 strain transformed with either the pGAD T7 MP clone or the pGBK T7 CP clone alone did not form colonies, ruling out the possi-bility of de novo activation of the reporter gene in the presence of the expressed proteins Similarly, untrans-formed AH109 S cerevisiae alone did not form any colonies (data not shown)

To identify the domain in MP that is involved in interaction with CP, MP mutant gene products obtained by PCR were cloned into the pGBK T7 vec-tor, and the mutants were tested for their interaction with CP expressed from the pGAD T7 vector Fig 8 shows that all the mutants exhibited positive Y2H interaction with CP The interaction between ND16 and CP (third row from top, Fig 8) was observed for growth under medium stringency conditions (–Leu⁄ – Trp⁄ –His ⁄ +a-X-Gal), but no interaction was observed under high stringency conditions (–Leu⁄ –Trp ⁄ –His ⁄ – Ade) For ND35 (fourth row from top in Fig 8A,B), the level of interaction was comparable to that between MP ND16 and CP pGAD T7 CP and pGBK T7 ND49 clones co-transformed into AH109 cells only formed white colonies in –Leu⁄ –Trp ⁄ – His⁄ +a-X-Gal plates (fifth row, column 4, in Fig 8A,B) These results show that the interaction of

A

B

Fig 7 Mapping of the SeMV MP domain responsible for interaction

of MP with NV (A) Purification of GST–MP deletion mutants GST–MP

deletion mutants were over-expressed in E coli BL21 (DE3) and

purified using glutathione affinity chromatography The Coomassie

brilliant blue-stained gel after 12% SDS ⁄ PAGE of purified GST–MP

deletion mutant proteins is shown (B) ELISA-based protein–protein

interaction assay between NV and GST–MP deletion mutants ELISA

plates coated with NV (P1) and blocked with 10% skimmed milk in

PBS and were incubated with GST–MP deletion mutants (P2) The

remaining stages of the reaction were performed as described in the

experimental procedure section using anti-MP polyclonal antibody

(pAb to P2) (hatched bars) Subsequent NV-coated wells were

incu-bated with GST and probed using anti-GST polyclonal antibody to rule

out the possibility of GST–NV interaction (white bars) Experimental

steps and controls are indicated in the figure The absorbance at

450 nm for each of the conditions is shown The percentage

decrease in absorbance for the mutants as compared to GST–MP

and NV interaction is indicated above the bars.

MP with CP is greatly reduced by deletion of 49

amino acids from the N-terminus C-terminal deletions

of MP (CD3, CD19 and CD38) (last three rows in

Fig 8) had a minimal effect on the interaction between

MP and CP However, none of the deletion mutants

grew on –Leu⁄ –Trp ⁄ –His ⁄ –Ade plates, indicating that

there was some loss of interaction (rows 3–8, column

5, in Fig 8A,B)

b-galactosidase assay with

ortho-nitrophenyl-b-galactopyranoside as substrate

Transformed colonies that showed a positive Y2H

interaction were grown in liquid culture (–Leu⁄ –Trp ⁄ –

His medium), and were assayed for b-galactosidase

activity to validate and quantify the results of the

two-hybrid interactions A single colony was picked from

each of the SD plates, and the b-galactosidase assay

was performed as described in Experimental

proce-dures The results are presented as a percentage of

arbitrary units of b-galactosidase activity (values

indi-cated on the top of each bar) relative to that obtained

with transformants expressing p53 and T-antigen

(100%) Values are the means of at least three separate

experiments (Fig 9A) There was no difference in the

b-galactosidase activity between transformants express-ing CP with MP, ND16, CD3, CD19 or CD38 How-ever, an appreciable decrease in activity was seen in transformants expressing CP with ND35 or ND49 Interaction between the SeMV MP and CP resulted in 71% activity compared with the interaction between p53 and the T-antigen (100%) The interaction between the DN49 mutant MP and CP resulted in 27% activity This corresponds to a reduction in activ-ity of 60% compared with interaction of the wild-type SeMV MP and CP This reduction in interaction could also be due to a difference in the level of expression of the interacting proteins To rule out this possibility, the levels of all the bait and prey proteins were quanti-fied by ELISA The CP expressed from pGADT7 vec-tor has a haemagglutinin tag fused to its N-terminus, and the MP and the mutants expressed from pGBK T7 have cMyc epitope tags The co-transformed colonies were grown overnight in 5 mL of appropriate selection medium, and cells were lysed to release the proteins ELISA was performed with the total lysate using haemagglutinin polyclonal antibody or cMyc monoclonal antibody at 1 : 10 000 dilution (Fig 9B) All the proteins were expressed at comparable levels Thus the drastic reduction in interaction of ND35 and

Fig 8 Y2H interaction between MP and CP (A) pGBK T7 (MP, MP deletion mutants and p53) and pGAD T7 (CP and T Ag) clones were transformed in pairs into the AH109 strain, plated onto –Leu⁄ –Trp SD transformant selection plates, and incubated for 96 h Colonies that grew were marked, and replica-plated onto various nutritional marker SD plates with various stringencies for reporter gene expression To assess a-galactosidase activity, colonies were plated onto SD plates containing a-X-Gal (B) Schematic representation of the results in (A).

ND49 with CP is because of deletion of the interacting

domain

Discussion

MPs are a diverse group of non-structural proteins of

plant viruses that are involved in the spread of infection

from cell to cell and systemically within the host plant [11] The present study comprised biochemical characterization of the MP encoded by ORF1 of SeMV, a member of the genus Sobemovirus Analysis

of the deduced amino acid sequence of the SeMV MP showed that it is predominantly an a-helical protein It has a C-terminal nucleic acid binding domain and a predicted phosphorylation site as expected of a protein involved in viral movement (Fig 1)

The interaction between MPs and virus- or host-encoded ancillary proteins is important for transport

of the viral genome from one cell to another The results presented here clearly show that the purified SeMV MP interacts with NV and CP, suggesting that SeMV might belong to the class of viruses that require

MP and NV⁄ CP for cell-to-cell movement

An inherent characteristic of MPs is their ability to interact with plasmodesmata and components of the cellular vasculature Hence, they tend to form inclusion bodies when expressed in vitro [36–38] The rMP over-expressed in E coli was also present in the insoluble fraction and was purified under denaturing conditions (Fig 2), but could be successfully refolded Upon denaturation with 8 m urea, in addition to the shift of the fluorescence emission maxima from 345 to 365 nm due to exposure of tryptophan residues, an additional broad peak at 305–315 nm was observed There is a single tryptophan (position 84) and eight tyrosines in the SeMV MP It is possible that the fluorescence emission of these tyrosines is quenched by energy transfer to tryptophan or charged amino groups or protonated carboxylates in their vicinity in the refolded protein However, upon denaturation, fluorescence due

to tyrosine is observed as a broad peak at 305–315 nm [32,39]

rMP eluted in the void volume when analysed by size-exclusion chromatography, suggesting that the refolded MP formed large oligomers (data not shown) Most MPs form soluble aggregates, probably because

of their inherent ability to form M complexes or tubules for transport across plasmodesmata In vitro, some MPs are known to form heteromeric complexes with various cytoskeletal elements and host factors such as the major nucleolar protein fibrillarin [40] Together with the MP, the CP plays a pivotal role

in cell-to-cell movement of certain viruses [3] In order

to examine interactions between the CP and the MP,

in vitro and ex vivo studies were performed The inter-action of rMP with NV was dependent on the concen-tration of rMP (Fig 5A) However, the stoichiometry

of interaction could not be estimated as the refolded rMP formed soluble aggregates Also, as the NV was immobilized on ELISA plates, not all the sites would

A

B

Fig 9 Quantification of the MP–CP Y2H interaction by

b-galactosi-dase assay and estimation of the level of protein expression (A) A

b-galactosidase assay of transformed colonies that showed positive

Y2H interaction was performed as described in Experimental

proce-dures The results are presented as a percentage of arbitrary units

of b-galactosidase activity (values are indicated on the top of each

bar) relative to the interaction between p53 and T-antigen (100%).

Values are means of at least three separate experiments (B) Direct

antigen-coating ELISA for estimation of the interaction between CP

and MP or its deletion mutants Total protein isolated from AH109

cells transformed with pGBK T7 MP or MP deletion clones and

pGAD T7 CP was coated on to ELISA plates The amount of MP

and the deletion mutants was quantified using cMyc monoclonal

antibody (open bars) The amount of CP was estimated using

hae-magglutinin polyclonal antibody (closed bars).