Indian Journal of Biochemistry & Biophysics Vol. 38. October 200 1. pp. 342-347

Purification and properties of antiviral proteins from the leaves of Bougainvillea xbuttiana l

Sneh Narwal, A Balasubrahmanyam, M L Lodha* and H C Kapoor Divi sion of Biochemi stry. Indian Agri cultural Research Institute, New Delhi 110 0 12, Indi J

Receil'ed 9 October 2000; revised alld accepted 18 April 2001

A non-phytotox ic. res istance inducing. proteinaceous ant iviral principle was purified by ammoniulll sulphate frac tionation. ion exchange chromatography and gel filtrati on from the leaves of BOllga ill villea xburtialla . It imparted res istance aga inst tobacco mosa ic vi rus (TMV) and sunnhemp rosette virus (SRV) in their respective test. hosts viz. Nicotialla gilltillosa. N. !abaclIlIl val'. Samsun NN, and Cyall/opsis letragolloloba, respecti vely. The purified principle eluted as a sin gle peak upon gel filtrati on. but ex hi bi ted two polypeptides on SDS-PAGE with M, 28,000 and 24,000. The two polypeptides were found to be highl y basic. ri ch in lys ine with pi around 10.0 and 10.5 , respectively. Since this principle effected loca l lesion inhibition in both treated and untreated top leaves of test host, it might be acting in the init ial stages of virus infection as a systemic inducer.

Plants bel onging to centrospermae are known to contain substances. which inhibit virus infection '·

4.

Presence of antiviral principles has been reported from a large number of plant species, however, on ly a few of the inhibitory substances have been purified and characterized. Still fewer have been studied for their mode of action during virus infection. Predominantly, the antiviral property has been found to be assoc iated with proteins, with or without sugar moiety. These subs tances with antiviral activity impart res istance either solely at the site of application (localized res istance) or at remote sites as well (systemic res istance), when applied at least sometime prior to vi rus challengeS. Recently , the plants containing these antiviral principles have been recognized as potent source of the non-viral genes for incorporating viral resistance into the plants by

. f . 6·8 genetic trans ormatton . Leaves of BOllgainvillea have been reported to

contain a virus inhibitory principle which is effective against a number of viruses'!· " . Out of four cultivated species of Bougainvillea (B. spectabilis, B. glabra, B. perL/viana, and B. xbuttiana) , only two species i.e. B. spectabilis and B. glabra l

•12 have been tested for

antiviral activity . Antiviral proteins have been isolated from the leaves ' 3 and roots l4 of B. spectabilis.

Aut hor for correspondence: Ema il : mllbio@ iari .e rnet.in : Fax: 91-0 I 1-5766420 I part of Ph.D Thesis submitted by SN to the Post­Graduate School, Indian Agricultural Research Institute, New Delhi .

We describe here the purification and chemical properties of two antiviral proteins from the leaves of B. xbuttiana whicll induce resi stance against TMV in Nicotiana tabacu/J'l var. Samsun NI'\ and SRV in Cyamopsis tetragolloloba.

Materials and Methods

Vi rus inoculum Cultures of tobacco mosaic virus (TMV) and

sunnhemp rosette virus (SRV) were maintained by regular passage in their systemic hosts Nicotiana tabacw/l L. var. NP33 and Cratolaria juncea L. , respectively. Virus inoculum was prepared by macerating the leaves show ing characteristic symptoms with O.IM sodium phosphate buffer (pH 7.2) in the ratio of 1:5 (w/v) . The pulp was squeezed through two layers of muslin cloth, and the filtrate was centrifuged at 12,OOOxg for 15 min. The supernatant was diluted with the same buffer to obtain countable number of lesions on the leaves of the test host plants.

Bioassay Nicotiana glutinosa and N. tabacum var. Samsun

NN were used as local lesion hosts for TMV and Cyalllopsis tetragol1oloba for SRV for conducting bioassay of the putative antiviral principle. All the test host plants were grown in plastic pots in insect free glass house. These virus-host combinations were used depending on season and availability of test plants. Plants of more or less same height. age and vigour

NARWAL el III.: PURIFICATION AND PROPERTI ES OF ANTIVIRAL PROTEINS FROM B. XBUITIANA 343

were selected for the experiments. For each treatment, three plants with 3-4 leaves of almost equal size were selected. Bioassay was performed as described previousli 5

.

The per cent inhibition of TMV or SRV infection was calculated by the formula

Per cent inhibition = ((C - T)/C) x 100

Where, C is the average number of les ions on control leaves and T is the average number of lesions on treated leaves.

Screening Bougainvillea species for virus inhibitory activity

Two cultivars each of four Bougainvillea species viz., B. spectabilis, B. glabra , B. peruviana and B. xbuttiana were tested for the presence of anti vital activity. For screening purpose, leaf extracts were prepared in di stilled water. Since the highest activity was shown by B. xbuttiana species, it was selected for further studies.

Purification of antiviral principle The procedure as described by Balasubrahmanyam

et al 16 after slight modification was followed. The leaves of B. xbllttiana cv. Enid Lancester were

washed with distilled water and dried at 40±2DC. The dried leaves were homogenized with 5-6 volumes of ex traction buffer (0.1 M 'sodium phosphate buffer, pH

7.0 containing 10mM ~-mercaptoethanol) and 20mg of polyvinylpolypyrro lidone (PVPP, Sigma) in a Waring blender. The pulp was squeezed through two layers of muslin cloth, and the filtrate was clarified by centrifugati on at 12,000xg for lOmin. The clear supernatant (crude extract) was used for the purification of antiviral principle.

Al/ll/loniul/I sulphate fra ctionation Initially the erude extract was subjected to different

saturation levels of ammonium sulphate in sequential manner i.e. 0-25, 25-90 and 90-100% . At each step, precipitated proteins were pelleted by centrifugation at 12,000 x g for 10 min and the pellets were suspended in minimum amount of sodium phosphate buffer (20mM, pH 6.2) containing lOmM NaC\. All the fractions were di alysed against the same buffer for

24hr with three changes at 4DC. Dialysed fraction s were tested for inhibitory activity.

DEAE-Celllllose chromaTOgraphy The active 25 -90% ammonium sulphate fraction

(1330mg) was loaded onto the DEAE-Cellulose

column equilibrated with buffer-A (20mM sod ium phosphate buffer, pH 6.2 containing 10 mM aC I) and the unadsorbed proteins were washed out with the column buffer till no more proteins were detected in the eluent. Adsorbed proteins were eluted with 0.5 M NaCI in the same buffer. Both adsorbed and unadsorbed fractions were tested for antiviral activity. The unadsorbed fraction showing inhibitory activity was used for cation exchange chromatography.

CM-Sepharose chromatography The active unadsorbed fraction (240mg) from

DEAE-Cellulose column was loaded onto the CM­Sepharose column (35 x 1.0 cm) equilibrated with buffer-B (20mM sodium acetate buffer, pH 5.2 containing 10mM NaCI and 0.0 I % sodium az ide). The column was washed with 4-5 bed volumes of equilibration buffer. Adsorbed proteins were eluted with a step gradient of NaCI (0.1 M , 0.2M, 0.3M. OAM and 0.5M) in equilibration buffer. At each step, 4-5 bed volumes were used and 3.75ml fractions collected on LKB fraction collector system at a flow rate of 30mllhr. Absorbance of each fraction was determined at 280nm. Fractions corresponding to different peaks were pooled and tested for antiviral activity.

Size-exclusion chrol/latography on Superose- 12 and Sephadex G-75 colunllIS

Active peak fractions were concentrated on sucrose

pad at 4 DC and loaded (2.3mg) onto the Superose- 12

column (45 x 1.0 cm) equilibrated with buffer-C (20mM sodium acetate buffer, pH 5.2 containing 0.2M NaCI and 0.0 I % sodium azide). Elution was done with the same buffer at a flow rate of 15mllhr and 1.6ml fractions were collected. Absorbance of all fractions was measured at 280nm. The fractions corresponding to different peaks were pooled and tested for antiviral activity.

Active protein fractions from CM-Sepharose column were also loaded on Sephadex G-75 column (61 x 1.5cm) equilibrated with 20mM sodium acetate buffer (PH 5.2, containing 0.01 % sodium azide). Elution was done with the same buffer at a flow rate of 15mllhr and pooled fractions for different peaks were tested.

Protein estimation

Protein concentration at each step of purification was determined by the method of Lowry el al. 17 with bovine serum albumin as standard.

344 INDIAN 1. BIOCHEM. BIOPHYS .. VOL. 38, OCTOB ER 2001

SDS-polyacrylamide gel electrophoresis Di scontinuous SDS-PAGE was carried out in 12%

separating gel with a 5% stacking gel according to Laemmli I 8

. The finally purified proteins were also analysed on 10-20% gradient gel. Proteins were visualized by staining with 0 . 1 % Coomassie brilliant blue R-250.

Moleclilar weight determination The molecular weights under denaturing conditions

were determined on 10-20% gradient gel by SDS­PAGE using following markers from Sigma (Dalton mark VI): bovine serum albumin (66,000), egg albumin (45 ,000), pepsin (34,700) , trypsinogen (24,000), lactoglobulin (18,400) and lysozyme (14,300) .

Carhohydrate estimation Presence of carbohydrates in the final Superose-I2

fraction was detected after dialysis of sample against buffer-C by Moli sch test and periodic acid-Schiff's reagent test I9 using ovalbumin as positive control and BSA as negative control. Quantitative estimation of total neutral sugars was done by phenol-sulfuric acid method20 using glucose as standard.

I soelectric focussing pi of the purified proteins were determined under

non-denaturating conditions by isoelectric focussing in 5% horizontal polyacrylamide slab gel containing carrier ampholytes (PH 3.0-10.5) as described by Righetti and Chillemi21.

Amino acid analysis Two protein bands in the final fraction were eluted

out separately from the SOS-polyacrylamide gel and used for amino acid analysis. Proteins were hydrolysed with 6N HCI in sealed and evacuated

tubes at 110°C for 24hr. Hydrolysed samples were derivatized with phenylisothiocyanate (PITC) and analysed by HPLC (Pico-Tag-Amino acid analysis system, operators manual, Waters, USA).

Systemic effect To study the property of resistance induction at

remote or untreated site, the basal leaves of the test host plants were treated with purified protein as described earlier. Then the virus was inoculated in the treated basal and untreated remote site leaves after different time intervals (0, 2, 6, 12, 24, 32 and 48hr). After development of the symptoms, per cent inhibition was calculated in each case.

Results and Discussion Bougainvillea xhuttiana (cv. Enid Lancester)

showing comparatively higher antiviral activity out of four species tested was selected for the purification and characterization of antiviral principle. Minor differences were observed in the inhib itory activity of leaf extracts prepared in buffers with the pH range o f 5 .2-8.0 (data not shown). Therefore, further extraction of antiviral principle was carried out with the buffer having pH 7.0.

Virus inhibitory substance from the leaves of B. xhutfiana was purified by conventional biochemical techniques. The antiviral principle was extracted from dried leaf material with 0.1 M sodium phosphate buffer (PH 7.0). The bulk of the activity was recovered from 25-90% ammonium sulphate fraction. This fraction after dialysis was subjected to DEAE­Cellulose chromatography. The unadsorbed fraction with most of the antiviral activity was then subjected to CM-Sepharose chromatography and the adsorbed proteins were eluted with a step gradient of NaCI. The peaks obtained with O.IM NaCI gave negligible activity. Peak-II obtained with 0.2M NaCI elution gave maximum inhibitory activity. This peak after concentration was subjected to ge l filtration on Superose-12 column. One major peak showing antiviral activity was obtained after gel filtration (Table I).

All the fractions starting from crude extract were subjected to SOS-PAGE (Fig. I). The tinal Superose-

12 fraction exhibited two bands. Elimination of 0-mercaptoethanol from the sample buffer did not make any difference. Molecular sieving on Sephadex G-75 also could not separate the two polypeptides. In case of Celosia cristata I6

, electropho resis of purified inhibitor exhibited two closely separated bands, one of which was predominant at pre-flo wering stage (CCP-25) and the other at post-flowering stage (CCP-27) of the plant, and they were suggested to be isoforms. Bolognesi et al. 13 also observed two polypeptide bands from single peak fractions in case of Basella ruhra and were unable to separate them by various non-denaturing chromatographic techniques. Based on the amino acid sequence, they suggested that the two proteins could be isoforms.

The molecular weights of two finaIIy purified antiviral proteins, as determined by 50S-PAGE were 33,000 and 28,000. But, electrophoresis on 10-20% gradient gel (Fig. 2) exhibited the molecular weights of 28,000 and 24,000, based on which, these Bougainvillea xhuttiana proteins are henceforth referred

NARWAL el al .: PURI FICATION AND PROPERTI ES OF ANTIVIRAL PROTEINS FROM B. XBUTTIANA 345

to as 28 kDa and 24 kDa proteins, respectively. This type of anomalous behaviour on polyacrylamide gel electrophoresis, is usually shown by glycoproteins as has been observed and di scussed in case of C.

Table I - Purification of anti viral protein from Bougainvillea xblilliana cv. Enid Lancester leaf extract

[Data refers to 60g of dry leaf materi al. At each step of purifi cation, the protein frac tions were tested for anti viral activity. 3-4 leaves of three C. lelragonoloba plants were treated with the prote in fract ion. After I hr. the leaves were washed and inocul ated with SRVI

Protein frac tion

Crude extract Ammonium sulphate fraction (25-90%) DEAE-Cellulose (unadsorbed) CM-Sepharose

O. IM NaC I Peak-I Peak- II Peak-Ill 0.2M NaC I Peak-I Peak-II Superose-12

0 b k Do

66 ·0-

1.5'0-31. ·7 -

21.·0 -

, B·t. --

tt. ·] -

Total protein

c

( mg)

3345.00 1433.60

245.7

2.01 7.29 6.88

12.90 2.43 1.552

d

..... e

Pro te in % concentration inhibition

(mg/ml )

15.00 99.56 10.24 98.68

1.17 100.00

0. 105 26.00 0.380 34.80 0. 190 33.00

0.335 61.15 0.095 96.94 0.080 96.50

\) h

---

Fig. I-SDS-PAGE (12%) analysis of protein fractions obtained at different stages of purification. [Lanes (a) and (i), molecular weight markers; lane (b), crude extract; lane (c), 25-90% ammonium sulphate fraction; lane (d), DEAE-Cellulose unadsorbed; lanes (e) and (f), Peak-I and Peak-II (0.2M NaCI elution) from CM-Sepharose; lane (g), Superose-12 fraction and lane (h), Superose-1 2 fraction without !3-mercaptoethanol. The proteins from these fractions were precipitated with ice cold TCA (10% final concentration) for 15 min in freezer and then centrifuged. The pellet was washed with ethanol: ether (1:1) and resuspended in sample buffer].

a b kDa

66·0-

l. 5·0-

24'0-

1 ",3-

Fig. 2-SDS-PAGE analysis of 28 kDa and 24 kDa proteins on 10-20% gradient gel. [Lane (a), molecular weight markers and lane (b), Superose-12 fraction].

cristata l 6• Both the proteins tested positi ve for

glycoprotein staInmg by periodic acid-Schiff s reagent test (Fig. 3), and the total neutral sugar content of the final Superose-12 fraction was 3.5%. Antiviral proteins isolated from a number of other plants like Boerhaavia dif.{usa22

, Dianthus caryophyllui3 and Celosia cristata l6 are also glycoproteins containing varying amounts of total neutral sugars. However, it differs from the antiviral protein purified from Chenopodium album24 in this property .

Both the proteins contained very high percentage of lysine (16.73% in 28 kDa protein 39.10% in 24 kDa protein), however, the other two basic amino acids, arginine and histidine were present in less amounts « 1.0%) (Table 2). A general trend observed for almost all virus inhibitors is the presence of alanine in very high amounts and histidine in very low amounts. The basic nature was further confirmed by their very high pI values of -10.0 (28 kDa) and -10.5 (24 kDa), also suggesting that these could be two

346 INDI AN 1. BIOCHEM. BIOPHYS .. VOL. 38. OCTOBER 2001

a b c

Fig. 3-Pcriodi c acid-SchifF's reagent staining of Superose-12 fraction. [Lane (a), Superose-12 fracti on; lane (b) . positive cont ro l. ova lbumin and lane (c). negative control, bovine serum albumin].

di fferent proteins. The properties of the two proteins are sim il ar to that of Mirabilis antiviral protein25

,

pokeweed antiviral protein26 and Yucca recurvifolia protein27 in respect of being lysine rich, basic proteins with pI greater than 9.5.

Results presented in Table 3 indicate the poss ibility that the purified antiviral proteins from B. xbuttialla cou ld induce systemic resistance. The per cent inhibition of virus induced lesions reached maximum 24hr after protein treatment in both treated and untreated top leaves, however, the extent of inhibition was more (27 %) in treated leaves. Similar observations have been made with Mirabilis antiviral

·?8 I f P d I protelll- . n case 0 seu erant 1emum atropurpureul1I29

, the maximum inhibition was observed between 24 and 48hr after the treatment in untreated top leaves. However, in case of Clerodendrulll inenne, the induced resistance was evident 4hr onwards and persisted upto 48hr at both treated as well as untreated remote sites5

. In contrast, the resistance induced by B. xbutliana proteins was observed to increase only up to 24hr and thereafter decreased sharply at both the sites.

Table 2-Amino acids present in the two B. xblllliall(l anti viral proteins"

The two proteins after elution from the gel were hydrolyzed at I lQoC for 24 hr, deriv atized with PITe and then subj ected to HPLC analysis]

Ami no acid Mole % in 28 kDa Mole % in 24 kDa protein protein

Aspartate 0.40 0.28 Glutamate 0.33 0.24 Serine 0. 16 nd Glycine 5.85 5.17 Hi stidine nd 0.27 Argini ne 0.65 0.55 Threonine 2.48 2.55 Alanine 69.60 32.56 Tyros ine 0.54 0.39 Cysteine nd 17.92 Lysine 16.73 39.1 0 Tryptophan* 3.2 1 2.95

*Determ ined spectrophotometrica ll y by the method of Spies and Chambers. nd = not detected

Table 3-Systemic inJuced resistance against SRV infection in Cyalllopsis lelragolloloiJa by the purifi ed inhibitor

[The values given represent the average of the data collected from three plants. Zero hour treatment represents virus inoculation immediately after protein treatment. Treatmenl with the purifi ed inhibitor was done on two basa l leaves of C. l erragolloloba plants. After different lime interval s, SRV inocul ations were done on all (two basal and two top) leaves]

Virus inoculation time after Per cent inhibition protein treatment of basal leaves Treated Untreated

(hours) basal remote leaves site leaves

0 95 .00 0.0 2 94.25 0.0 6 94.25 37.00 12 93.50 55.50 24 96.00 70.00 32 57.50 29.50 48 44.50 5.62

Development of systemic resistance is a host­mediated phenomenon. However, how exactly it is induced is not clearly known. It may involve the synthesis of new proteins in the host in response to antiviral protein treatment, which in turn , may be acting as true virus inhibitor. Further, this response may be capable of translocation to top leaves as observed in case of B. dijfusa30 and Cleroclclldrum aculeatw1l31

• Some of the antiviral protei ns discovered so far including the one from B. xbuttiana32

, were observed to be ribosome inactivating proteins (RIP). However, recent studies show that RIP property by

NARWAL el al .: PURIFICATION AND PROPERTI ES OF ANTIVIRAL PROTEINS FROM B. XBU7TIANA 347

itself is not responsible for antiviral action. It may help generate a signal that renders the plant resistant to virus infection33

.

Acknowledgement The seni or author (SN) is thankful to the Director,

Indian Agricultural Research Institute, New Delhi fo r the award of Senior Research Fellowship during the course of thi s study. The support provided by the ICAR Centre of Advanced Studies in Biochemistry is gratefull y acknowledged.

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