discontinuous gradient of NaCl 0.1 and 0.2 M prepared in the same buffer. About four to five
bed volumes of buffer were used at each step and fractions 3.0 ml were collected on an LKB frac-
tion collector system at a flow rate of 30 ml h
− 1
. Protein elution was simultaneously monitored at
280 nm and signals were traced on to a printer. Fractions falling within the protein peak were
pooled and tested for antiviral activity.
2
.
3
.
4
. Gel filtration chromatography The pooled protein sample 1.9 mg was either
lyophilized at − 20°C or concentrated in a dialy- sis bag on a sucrose pad at 4°C. Later it was
chromatographed on a Sephadex G-75 Pharma- cia Fine Chemicals, Uppsala, Sweden column
61 × 1.5 cm equilibrated with buffer C 20 mM sodium acetate, pH 5.2 containing 0.01 sodium
azide. The protein was eluted at a constant flow rate of 40 ml h
− 1
and fractions 3.1 ml were monitored at 280 nm. Fractions falling within the
protein peak were pooled and tested for antiviral activity.
2
.
4
. Protein and carbohydrate estimation Protein estimation was done by the Bradford
method [12] using bovine serum albumin BSA as a standard. The presence of carbohydrate was
tested by the Molisch test [13] and the estimation of total neutral sugars was done by the phenol-
sulfuric acid method [14].
2
.
5
. Protein characterization SDS-PAGE was performed in a 12 separating
gel according to Laemmli [15]. The proteins were also analyzed in a 10 – 20 linear gradient gel.
The gradient was prepared using an LKB gradi- ent maker. The proteins were visualised with
Coomassie blue or by silver staining [16]. Glyco- proteins were detected on polyacrylamide gels us-
ing the periodic acid-Schiff reagent PAS [17]. Ovalbumin was used as a positive control on the
gel, while BSA served as the negative control. The molecular weights of the purified proteins
were determined under native conditions by cali- brated
gel-permeation chromatography
using MW GF-70 kit contains aprotinin, 6.5 kDa; cy-
tochrome c, 12.4 kDa; carbonic anhydrase, 29 kDa and bovine serum albumin, 66 kDa Sigma
in a Sephadex G-75 column. The R
f
of molecular marker and purified proteins were plotted against
the molecular weight on a semi-log graph paper. The molecular weights of the purified proteins
were also determined under denatured conditions in a 12 polyacrylamide gel and a 10 – 20 linear
gradient gel by SDS-PAGE using MW SDS-70 kit.
2
.
6
. Stability to protease digestion The purified proteins, obtained by the above
mentioned purification procedure, were incubated at 37°C for 12 h with trypsin, chymotrypsin,
Staphylococcus aureus protease XVII-B or papain. The
incubation mixture
contained purified
protein and protease in a ratio of 25:1 ww in 50 mM Tris – HCl buffer, pH 6.8. The mixture
was then applied onto test plants to determine its antiviral activity. Both proteins, after incubation,
were also monitored by SDS-PAGE. The antivi- ral proteins were also denatured in Cleveland
buffer [18] and digested with each of the above proteases for 15 or 30 min and subjected to SDS-
PAGE.
2
.
7
. Effect on systemic hosts The purified protein was tested for its ability to
impart resistance in different systemic host-virus combinations. About 60 mg of purified protein in
1 ml was mixed with each of the purified viruses TMV, SRV and CRSV, and the mixture was
applied on carborundum dusted leaves of three test plants each of tobacco cv NP33, sunnhemp
and French bean, respectively. The symptoms were observed after 25-35 days of virus infection
in case of TMV and SRV, and after 6 days in case of CRSV. Back-inoculation or electron mi-
croscopy or both were carried out to detect the virus in the treated systemic host plant.
3. Results
Preliminary studies had shown that an extract of dry leaf material dried at 40 – 45°C of C.
cristata had antiviral activity comparable to that of fresh leaves. Therefore, leaves harvested at dif-
ferent growth stages of the Celosia plant were separately dried and were used to extract the
antiviral activity.
Table 1 Purification of antiviral protein from Celosia cristata
a
Test sample concentration mg ml
− 1
Fraction Percent of inhibition
b
Total protein mg 0.024
Crude extract 75.0
24.52 25–80 ammonium sulphate
18.80 0.117
95.0 0.164
95.0 DEAE-cellulose unadsorbed
9.30 CM-Sepharose purified
0.142 Peak I
13.5 4.49
0.125 1.98
97.5 Peak II
Peak III 2.50
0.175 −
5.9 0.060
1.55 98.0
Sephadex G-75 single peak
a
Data refers to 40 g of C. cristata dried leaf material.
b
Three to four well-developed leaves of four test plants of Nicotiana tabacum cv. Samsun NN, each for all sets, were treated with 2 ml of test sample. After 1 h the leaves were washed with distilled water and blotted dry. The leaves were dusted with
carborundum and inoculated with tobacco mosaic virus.
3
.
1
. Optimum pH for extraction Initial studies showed that the inhibitor ex-
tracted with 20 mM sodium acetate buffer, pH 5.2, exhibited maximum activity 95.6 local lesion
inhibition compared to that of other extracts made with different buffers of varying pH. Fur-
ther, the few lesions produced in this case were very small and not very well developed compared
to those in other treatments results not shown. Therefore, this buffer, with higher molarity 0.1
M and supplemented with 12 mM b-mercap- toethanol was used for extracting the antiviral
activity for subsequent purification process.
3
.
2
. Purification of inhibitor Upon stepwise ammonium sulphate precipita-
tion, most of the antiviral activity was recovered in the fraction precipitating between 25 and 80
ammonium sulphate. This pellet was redissolved in buffer A, dialyzed against the same buffer and
subjected to DEAE-cellulose chromatography. The antiviral activity that came through in the
void volume was further fractionated by CM-sep- harose cation exchange chromatography, using a
discontinuous linear NaCl gradient 0.1 and 0.2 M to elute bound proteins. The elution at 0.1 M
NaCl resulted in one protein peak, peak I, whereas 0.2 M NaCl gave rise to two peaks, peaks II and
III. Negligible amounts of protein were eluted at higher salt concentrations results not shown. Of
these, the peak II fractions exhibited maximum activity Table 1. The relevant pooled fractions
were subjected to size exclusion chromatography on Sephadex G-75. This resulted in a single
protein peak, which was subsequently found to possess the antiviral activity.
3
.
3
. Characterization of C. cristata anti6iral protein
3
.
3
.
1
. SDS-PAGE Electrophoresis of the Sephadex G-75 chro-
matography purified inhibitor under denaturing
Fig. 1. SDS-PAGE 12 of antiviral proteins of Celosia cristata at different stages of purification. Lanes a and h
carry molecular weight kDa markers, and lane b crude extract 30 mg, lane c 25 – 80 ammonium sulphate precipi-
tated 80 mg, lane d DEAE-cellulose unadsorbed 30 mg, lane e CM-sepharose purified 30 mg, lane f 2 and g 3.5
mg of Sephadex G-75 purified protein fraction, respectively. The proteins from these fractions were precipitated with ice
cold TCA 10 final concentration for 15 min in the freezer and the suspension was centrifuged. The pellet was washed
with ethanol:ether 1:1 to remove traces of TCA and resus- pended in Laemmli buffer before loading on to the gel.
Fig. 2. SDS-PAGE of antiviral proteins purified at different growth stages of Celosia cristata. Lanes b and c show 8
and 16 mg of antiviral protein, respectively, purified at pre- flowering stage, lanes e and f 7 and 15 mg of protein
purified at flowering stage, lane a 8 mg protein purified at post-flowering stage, and lane d molecular weight markers
as in Fig. 1. Protein in each sample was concentrated by TCA precipitation as described in the legend for Fig. 1.
ing, they were present in equal concentration when they were purified from the leaves harvested
at flowering stage. The leaves harvested at post- flowering stage 20 – 25 days after flowering, DAF
showed a prominent higher molecular weight polypeptide band Fig. 2. Both of these proteins
independently exhibited antiviral activity against TMV on N. tabacum Samsun NN.
3
.
3
.
2
. Molecular weight of anti6iral proteins Molecular weights M
r
of native Celosia antivi- ral proteins were determined by calibrated gel
permeation chromatography. A Sephadex G-75 column was calibrated by using the Sigma MW-
GF-70 marker kit. Proteins purified from different lots showing a single polypeptide on SDS-PAGE
were passed through the calibrated Sephadex G-75 column separately. One of them showed a molecu-
lar weight of 33 kDa and other showed a molecu- lar weight of 37 kDa. However, on SDS-PAGE
the M
r
of the higher mobility species was 33 kDa, while for the second polypeptide, it was slightly
different with a M
r
of 35 kDa Fig. 2. Since the estimation of M
r
of glycoproteins by gel fitration or non-gradient SDS-PAGE may give erroneous
results [19,20], electrophoresis was also carried out in a 10 – 20 linear gradient gel to confirm the
above data. The results produced M
r
of 25 and 27 kDa, respectively, for the aforesaid polypeptides
Fig. 3. Therefore, these proteins will be referred as CCP-25 and CCP-27, respectively CCP stands
for C. cristata protein.
3
.
3
.
3
. Presence of carbohydrates Both the 25 and 27 kDa Celosia antiviral
proteins stained positively for glycoproteins on polyacrylamide gels when PAS reagent was used
not shown. Ovalbumin, which served as a posi- tive glycoprotein control, also stained pink, while
BSA, which is not a glycoprotein, did not stain. Total neutral sugar contents, as determined by the
phenol-sulfuric acid method were 26 and 28, respectively not shown.
3
.
3
.
4
. Effect of dilution, temperature and exogenous proteases
CCP-25 and CCP-27 obtained after Sephadex G-75 chromatography were used for these studies.
Various concentrations of the two proteins, rang- ing from 10 – 150 mg ml
− 1
as determined by the Bradford method [11], were applied on tobacco cv.
Samsun NN plants to test for the resistance re-
Fig. 3. Linear gradient 10 – 20 SDS-PAGE of antiviral proteins purified at different growth stages of Celosia cristata.
Lane a molecular weight markers as in Fig. 1; lane b 4 mg of CCP-25 purified at pre-flowering stage; lane c CCP-25
and CCP-27 8 mg purified at flowering stage; and lane d 4 mg of CCP-27 purified at post-flowering stage. Protein in each
sample was concentrated by TCA precipitation as described in the legend for Fig. 1.
conditions revealed a prominent band and a closely separated faint band. These bands were
consistently exhibited in fractions obtained after DEAE-cellulose chromatography Fig. 1. How-
ever, the concentration of each polypeptide varied with the growth stage of C. cristata plants. While
the lower molecular weight polypeptide predomi- nated at initial stages of plant growth pre-flower-
Fig. 4. SDS-PAGE analysis of CCP-25 treated with different proteases. Approximately 15 mg of CCP-25, after dissociation
with Cleveland buffer [17], were incubated with different proteases 0.6 mg for 15 or 30 min. After digestion, the
samples were made to 10 b-mercaptoethanol and 2 SDS, heated at 100°C for 2 min and electrophoresed on 15
polyacrylamide gel. Lane a untreated CCP-25; lanes b and c trypsin treated; lanes d and e chymotrypsin treated;
lanes f and g Staphylococcus aureus protease XVII-B treated; and lanes h and i papain treated CCP-25. The first
lane for each enzyme represents the treatment for 15 min and the second one represents the same for 30 min.
lose the ability to impart resistance not shown. CCP-25 and CCP-27 were tested for their sensi-
tivity to proteases by incubation with trypsin, chymotrypsin, S. aureus protease XVII-B and pa-
pain. Application of protease-treated CCP-25 and CCP-27 on tobacco cv. Samsun NN, did not result
in any increase in lesion number. However, the proteases caused some reduction in TMV lesion
number themselves, when applied alone on test plants followed by challenge inoculation with
TMV. SDS-PAGE of native antiviral proteins in- cubated with the proteases for 24 h revealed that
their degradation was minimal, but after denatura- tion with Cleveland buffer [18] they were digested
to a large extent within 30 min of incubation treatment of CCP-25 with different proteases is
shown in Fig. 4. In contrast, BSA was completely digested when incubated either in native or dena-
tured form with each of these proteases not shown.
3
.
4
. Effect of purified proteins on systemic hosts Both CCP-25 and CCP-27 were independently
tested for their ability to induce resistance on systemic host plants. However, the results are pre-
sented only for CCP-25 in Table 2. In the case of tobacco-TMV system, symptoms appeared on
both control 26 days post-inoculation, pi and treated 35 days pi plants, albeit symptoms were
delayed on treated plants. Subsequent back inocu- lation on local lesion host N. tabacum cv. Samsun
sponse. CCP-25 inhibited lesion formation by more than 90 at a concentration as low as 20 mg
ml
− 1
, whereas CCP-27 exhibited the same re- sponse at a concentration of 30 mg ml
− 1
results not shown. Both CCP-25 and CCP-27 displayed
tolerance to temperature and remained entirely unaffected even after 10 min of incubation at
90°C. However, at around 95°C, they began to
Table 2 Effect of antiviral protein CCP-25 on different virus–systemic host combinations
a
Symptom Treatment
Systemic host–virus Lesion number upon back
Virus particles observed by EM
inoculation
b
9 S.E.M.
expression N. tabacum cv NP33,
Very high Control
Systemic mosaic 285 9 12.3
TMV 156 9 9.1
High Treated
Mosaic 87 9 7.2
High Crotalaria juncea, SRV
Control Systemic mosaic
Few 7 9 1.0
Nil Treated
Very high Control
Phaseolus 6ulgaris, Systemic mosaic
– CRSV
– Nil
Treated Nil
a
The purified protein CCP-25 60 mg ml
− 1
was mixed with the isolated virus in each case and applied on carborundum dusted lower leaves of respective systemic host plants. After 25–35 days of virus infection in case of tobacco mosaic virus TMV and
sunnhemp rosette virus SRV, and after 6 days in case of citrus ring spot virus CRSV, top leaves were harvested and analyzed for the presence of virus by back inoculation on respective local lesion hosts or by electron microscopy or both.
b
Mean number of lesions obtained on 12 leaves of four test plants.
Table 3 Effect of actinomycin D on antiviral property
a
Treatment Percent of
Average lesion number
b
9 S.E.M.
inhibition 162 9 9.4
Buffer control –
– 156 9 7.1
Actinomycin D 78.6
Positive control 34 9 3.2
Clerodendrum inerme extract ,
1:6 wv 149 9 11.4
CI+AMD 6.2
CI+AMD 12 h 47 9 5.1
70.4 after
CCP-25 92.4
12 9 2.0 30.4
CCP-25+AMD 111 9 12.7
42 9 4.4 73.5
CCP-25+AMD 12 h after
CCP-27 91.8
13 9 1.0 21.3
125 9 13.2 CCP-27+AMD
39 9 5.1 75.4
CCP-27+AMD 12 h after
a
Purified CCP-25 and CCP-27 30 mg ml
− 1
each were separately applied on leaves of tobacco cv. Samsun NN. In
one set, actinomycin D AMD, 20 mg ml
− 1
was applied immediately after the treatment, while in another set AMD
treatment was given after 12 h of CCP application. Challenge inoculations were made as usual. Control sets were treated
with buffer alone or with the AMD solution alone. Dry leaf extract of Clerodendrum inerme CI prepared with 20 mM
sodium acetate, pH 5.2 1:6, wv was used as a positive control [6].
b
Mean number of lesions obtained from 12 leaves of four test plants.
3
.
5
. Effect of actinomycin D on anti6iral property Both CCP-25 and CCP-27 imparted resistance
to tobacco cv. Samsun NN against TMV. This resistance could be largely inhibited when actino-
mycin D AMD, 20 mg ml
− 1
was applied immedi- ately after treating the plant with either CCP-25 or
CCP-27. When AMD was applied 12 h after CCP treatment, it failed to inhibit the resistance re-
sponse Table 3.
4. Discussion
