Báo cáo khoa học: Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins potx

Analysis of the
in planta
antiviral activity of elderberry
ribosome-inactivating proteins
Frank Vandenbussche
1
, Stijn Desmyter
1
, Marialibera Ciani
2
, Paul Proost
3
, Willy J. Peumans
1
and Els J. M. Van Damme
4
1
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium;
2
Dipartimento di Patologia
sperimentale, Universita
`
di Bologna, Italy;
3
Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit
Leuven, Belgium;
4
Department of Molecular Biotechnology, Gent University, Belgium
Although the type-2 ribosome-inactivating proteins (SNA-I,
SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are
all devoid of rRNA N-glycosylase activity towards plant

ribosomes, some of them clearly show polynucleotide–
adenosine glycosylase activity towards tobacco mosaic virus
RNA. This particular substrate specificity was exploited to
further unravel the mechanism underlying the in planta
antiviral activity of ribosome-inactivating proteins. Trans-
genic tobacco (Nicotiana tabacum L. cv Samsun NN) plants
expressing the elderberry ribosome-inactivating proteins
were generated and challenged with tobacco mosaic virus in
order to analyze their antiviral properties. Although some
transgenic plants clearly showed antiviral activity, no clear
correlation was observed between in planta antiviral activity
of transgenic tobacco lines expressing the different ribosome-
inactivating proteins and the in vitro polynucleotide–
adenosine glycosylase activity of the respective proteins to-
wards tobacco mosaic virus genomic RNA. However, our
results suggest that the in planta antiviral activity of some
ribosome-inactivating proteins may rely on a direct mech-
anism on the virus. In addition, it is evident that the working
mechanism proposed for pokeweed antiviral protein cannot
be extrapolated to elderberry ribosome-inactivating proteins
because the expression of SNA-V is not accompanied by
induction of pathogenesis-related proteins.
Keywords: elderberry; polynucleotide–adenosine glycosylase
activity; ribosome-inactivating protein; Sambucus nigra;
viral protection.
Ribosome-inactivating proteins (RIPs; EC 3.2.2.22) are
a heterogeneous family of structurally and evolutionary
related plant proteins sharing a common functional domain
that catalytically removes a specific adenine residue from a
highly conserved, surface-exposed stem-loop structure

found in the large rRNA of prokaryotic and eukaryotic
ribosomes [1,2]. At present, they are subdivided on the basis
of the structure of the genes and the corresponding proteins
into holo-RIPs and chimero-RIPs [3]. Whereas holo-RIPs
consist exclusively of a single catalytically active protomer
of either one (classical type-1 RIPs) or two smaller
polypeptide chains (e.g. maize RIP b-32), chimero-RIPs
are built up of chimeric protomers with an N-terminal
catalytically active domain arranged in tandem with a
structurally and functionally unrelated C-terminal domain
(classical type-2 and type-3 RIPs).
Biochemical and molecular studies have shown that the
elderberry tree expresses a complex mixture of type-2 RIPs
and/or lectins in virtually all tissues. In agreement with the
chronological order of their discovery, these Sambucus nigra
agglutinins (SNAs) are numbered SNA-I to SNA-V. The
first elderberry lectin was identified in bark tissue and
described as a NeuAc(a-2,6)Gal/GalNAc-specific agglutinin
(called SNA-I) [4,5]. Although already discovered in 1984,
SNA-I was recognized as a type-2 RIP only when the
corresponding gene was cloned in 1996 [6]. Besides SNA-I,
elderberry bark contains a second NeuAc(a-2,6)Gal/Gal-
NAc-specific agglutinin which shares 77% sequence simi-
larity with SNA-I (but has a different oligomeric
organization) and was called SNA-I¢ [7]. The second lectin
identified in elderberry bark was a GalNAc-specific agglu-
tinin, called SNA-II with no obvious relation to SNA-I [5].
However, molecular cloning of a GalNAc-specific elder-
berry bark type-2 RIP (called SNA-V) later revealed that
SNA-II consists of subunits that correspond to slightly

truncated B chains of this genuine type-2 RIP. Both SNA-V
and SNA-II are derived from a single precursor, through
differential processing [8]. It should be mentioned here that
SNA-V (or a very closely related paralog) was first described
by Girbes et al. [9] as nigrin b. After the identification of the
bark lectins SNA-I and SNA-II, two other elderberry lectins
called SNA-III and SNA-IV were isolated from seeds and
Correspondence to E. J. M. Van Damme, Department of Molecular
Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent,
Belgium. Fax: + 32 9264 6219, Tel.: + 32 9264 6086,
E-mail:
Abbreviations: PAG, polynucleotide–adenosine glycosylase; PAP,
pokeweed antiviral protein; PR, pathogenesis-related; RIP, ribosome-
inactivating protein; SNA, Sambucus nigra agglutinin; SNLRP,
Sambucus nigra lectin-related protein; TMV, tobacco mosaic virus.
Enzyme: Ribosome-inactivating protein, rRNA N-glycosylase
(EC 3.2.2.22).
(Received 16 January 2004, revised 24 February 2004,
accepted 27 February 2004)
Eur. J. Biochem. 271, 1508–1515 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04059.x
fruits, respectively. Molecular cloning revealed that SNA-
IV, which is the most abundant fruit protein, is a dimeric
GalNAc-specific lectin encoded by a truncated type-2 RIP
gene (with a major deletion comprising almost the whole A
chain) [10]. Besides the different SNAs, the bark of
elderberry contains an additional protein, SNLRP, which
is both structurally and evolutionary closely related to the
other elderberry type-2 RIPs but possesses a B chain which,
because of several amino-acid substitutions in the sugar-
binding sites, is devoid of carbohydrate-binding activity [11].

Initially, RIPs were thought to act exclusively on
ribosomes or rRNA through their rRNA N-glycosylase
activity. However, using a highly sensitive HPLC-fluores-
cence-based method, Barbieri et al. [12] showed that several
RIPs release more than one adenine residue from rRNA.
Moreover, they found that saporin L1, a type-1 RIP from
the leaves of soapwort (Saponaria officinalis L.), is capable
of removing multiple adenine residues from various nucleic
acid substrates including herring sperm DNA, mammalian
DNA, genomic viral RNA, rRNA and poly(A) [13–15].
Additional testing of 61 RIPs revealed that all of them
extensively deadenylate herring sperm DNA and that
several are active towards viral genomic RNA [16–20].
On the basis of these findings it has been suggested that
polynucleotide–adenosine glycosylase (PAG) activity may
be responsible for the potent antiviral activity of RIPs
(against both animal and plant viruses). However, it is
still unclear whether the results obtained in these in vitro
studies can be extrapolated to the complex environment of
acell[16].
Although the antiviral activity of RIPs against plant
viruses is well documented, the underlying mechanism(s) has
not yet been elucidated. In principle, three possible expla-
nations can be put forward [3,21]. First, RIPs may act
directly on the viral nucleic acids through their PAG activity.
Secondly, RIPs may act directly on the host by selectively
killing the infected cells, thus preventing the virus from
replicating and spreading to neighboring cells. Finally, RIPs
may act indirectly through activation of the plant’s defence
system. As most RIPs with a documented strong in planta

antiviral activity [e.g. pokeweed antiviral protein (PAP),
trichosanthin] are able to depurinate both viral nucleic acids
and plant ribosomes, it has been difficult to assess how the
PAG activity contributes to RIP-mediated protection. To
further unravel the mode of action of RIPs, the in planta
antiviral activity of a set of type-2 RIPs (SNA-I, SNA-V,
SNLRP) from elderberry (S. nigra L.) which are all devoid
of rRNA N-glycosylase activity towards plant ribosomes [9]
but strikingly differ from each other with respect to their
PAG activity towards tobacco mosaic virus (TMV) RNA,
was analyzed using a TMV/tobacco (Nicotiana tabacum L.
cv. Samsun NN) model system. In addition to the genuine
type-2 RIP, the elderberry lectin SNA-IV, which is consid-
ered a type-2 RIP without an A chain, was included in the
study as a negative control. A comparison of the protection
offered by the different ectopically expressed elderberry
RIPs did not reveal a clear correlation between in planta
antiviral activity and PAG activity towards TMV genomic
RNA. However, it is evident that the working mechanism
suggested for PAP cannot be extrapolated to elderberry
RIPs because expression of the latter is not accompanied by
induction of pathogenesis-related (PR) proteins.
Materials and methods
Plasmid constructions
All manipulations were performed according to standard
techniques [22]. The pGK vector was constructed by
replacing the b-glucuronidase gene from the plant transfor-
mation vector pGPTV-KAN [23] with the expression
cassette of the pFF19 vector [24]. The coding sequences of
the various RIPs/lectins were amplified by PCR to engineer

appropriate restriction sites. The restricted PCR products
were inserted between the cauliflower mosaic virus 35S
promoter and polyadenylation signal of the linearized and
dephosphorylated pFF19 vector. After confirmation of the
sequence by dideoxy sequencing [25], inserts were subcloned
into the expression cassette of the pGK plant transforma-
tion vector. The resulting plasmids, pGKsnaI,pGKsnaIV,
pGKsnaV and pGKsnlrp, were transferred into Agrobacte-
rium tumefaciens GV3101 by electroporation [26].
Transformation of
N. tabacum
Transformation of tobacco (N. tabacum L. cv. Samsun
NN) was performed using the leaf disc cocultivation method
[27]. Transgenic shoots were selected on Murashige-Skoog
medium supplemented with 0.1 mgÆL
)1
a-naphthalene
acetic acid, 1 mgÆL
)1
6-benzylaminopurine, 100 mgÆL
)1
cefotaxime, 100 mgÆL
)1
carbenicillin and 100 mgÆL
)1
kana-
mycin (Duchefa Biochemie BV, Haarlem, the Netherlands).
Transformed plants were kept in a culture room or a
greenhouse at 22 °C, 50% relative humidity, and a 16 h
photoperiod until use.

Molecular analysis of transformants
Total genomic DNA was isolated as described by Goode &
Feinstein [28]. The presence of the transgenes was investi-
gated with PCR using two internal primers derived from the
N-terminal and C-terminal sequence of the various RIPs/
lectins. Only the PCR-positive plants were further analysed
at the RNA and protein level.
Total RNA was isolated as described by Eggermont et al.
[29], dissolved in RNase-free water, and quantified spectro-
photometrically. Approximately 30 lgtotalRNAwas
denatured with glyoxal/dimethyl sulfoxide and separated
on a 1.2% (w/v) agarose gel. After electrophoresis, RNA was
capillary blotted on to Hybond-N
+
membranes (Amersham
Biosciences, Uppsala, Sweden). Membranes were first
probed with random-primer-labeled cDNAs encoding the
different RIPs/lectins or PR proteins (PR-1, PR-2, PR-3,
proteinase inhibitor II). Subsequently the membranes were
reprobed with a random-primer-labeled cDNA fragment
complementary to the 3¢ end of the tobacco 25S rRNA.
To estimate the expression levels of the different RIPs/
lectins, transgenic lines (T
1
generation) were analysed for
recombinant (r)RIP/lectin content by Western blot densi-
tometry. To minimize variation, 10 selfed plants of each line
were grown under identical conditions (22 °C, 50% relative
humidity, 16 h photoperiod). When plants reached the six-
leaf stage, the third and fourth leaf were pooled, lyophilized,

and ground using mortar and pestle. Total protein from
50 mg lyophilized leaf material was extracted in 100 m
M
Ó FEBS 2004 Antiviral activity of ribosome-inactivating proteins (Eur. J. Biochem. 271) 1509
Hepes (pH 7.6). The protein concentration was determined
using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA,
USA) using BSA as standard [30]. For Western blot analysis,
 2.5 lg(SNA-V),7.5 lg (SNLRP) or 15 lg (SNA-I, SNA-
IV) total protein was separated by electrophoresis on a 15%
(w/v) SDS/polyacrylamide gel and transferred to an Immo-
bilon-P membrane (Millipore, Bedford, MA, USA) using a
Trans-blot SD semidry transfer cell (Bio-Rad). Immuno-
detection of the recombinant proteins was performed as
described by Desmyter et al. [31] using affinity-purified
polyclonal rabbit antibodies raised against native SNA-I,
SNA-II or SNLRP as the primary antibody.
Purification of recombinant proteins
rRIPs were isolated using a combination of classical protein
purification techniques and affinity chromatography (except
for SNLRP, which possesses no sugar-binding activity). For
the purification of recombinant SNA-I, leaves of SNA-I
transformants were homogenized in a solution of 1 gÆL
)1
ascorbic acid using a Waring blender. After centrifugation
at 3000 g for 10 min, 1 gÆL
)1
CaCl
2
was added to the
supernatant, and the pH adjusted to 9.0 with 0.5

M
NaOH.
The extract was cleared by centrifugation at 8000 g for
10 min and filtered through Whatman 3MM filter paper.
The filtrate was subsequently adjusted to pH 2.8 with 0.5
M
HCl and loaded on to a column (2.6 cm · 10 cm; 50 mL
bed volume) of S Fast Flow (Amersham Biosciences,
Uppsala, Sweden) equilibrated with 20 m
M
acetic acid.
After loading, the column was washed with 20 m
M
acetic
acid until the A
280
fell below 0.01, and the bound proteins
were eluted with 300 mL 0.1
M
Tris/HCl (pH 8.7) contain-
ing 0.5
M
NaCl. This partially purified protein fraction was
adjusted to pH 7.0 with 0.5
M
NaOH and loaded on a
column (1.6 cm · 5 cm; 10 mL bed volume) of fetuin–
Sepharose 4B equilibrated with 0.2
M
NaCl. After the

columnhadbeenwashedwith0.2
M
NaCl, the bound lectin
was desorbed with 20 m
M
1,3-diaminopropane (pH 9.0)
andstoredat)20 °C until use.
For the purification of recombinant SNA-IV and SNA-
V, leaves of SNA-IV and SNA-V transformants were
homogenized in 20 m
M
citric acid (pH 3.0) using a Waring
blender. The crude extract was cleared by centrifugation at
3000 g for 10 min and filtered through Whatman 3MM
filter paper. (NH
4
)
2
SO
4
was added to a final concentration
of 1.5
M
and the pH adjusted to 7.0 with 0.5
M
NaOH. The
extract was then applied to a column (2.6 cm · 10 cm;
50 mL bed volume) of galactose–Sepharose 4B equilibrated
with 1.5
M

(NH
4
)
2
SO
4
. After the column had been washed
with 1.5
M
(NH
4
)
2
SO
4
until the A
280
fell below 0.01, bound
proteins were desorbed with 0.2
M
galactose in 1.5
M
(NH
4
)
2
SO
4
. The affinity-purified proteins were loaded on
a column (1.6 · 10 cm; 20 mL bed volume) of phenyl-

Sepharose (Amersham Biosciences) equilibrated with 1.5
M
(NH
4
)
2
SO
4
. After the column had been washed with 1.5
M
(NH
4
)
2
SO
4
, the protein was desorbed with 20 m
M
1,3-
diaminopropane (pH 9.0). Fractions (2.5 mL each) were
collected and analysed by SDS/PAGE. Peak fractions
containing the RIPs/lectins were pooled and stored at
)20 °C until use.
For the purification of rSNLRP, leaves from SNLRP
transformants were homogenized in 50 m
M
acetic acid
using a Waring blender. After adjustment of the pH to 3.0
with 0.5
M

HCl, the homogenate was centrifuged at 3000 g
for 10 min and filtered through Whatman 3MM filter
paper. The cleared filtrate was then loaded on a column
(2.6 cm · 10 cm; 50 mL bed volume) of S Fast Flow
(Amersham Biosciences) equilibrated with 20 m
M
acetic
acid. After the column had been washed with 100 mL
25 m
M
sodium formate (pH 3.8), the bound proteins were
desorbed with a linear gradient (500 mL) of increasing
NaCl concentration (from 0 to 0.5
M
). Fractions (5 mL
each) were collected and analysed by SDS/PAGE. Peak
fractions containing SNLRP were pooled, concentrated by
freeze-drying, and loaded on a column (2.6 cm · 70 cm;
350 mL bed volume) of Sephacryl 100 equilibrated with
10 m
M
Tris/HCl (pH 7.5) for gel filtration. Finally, frac-
tions (2.5 mL each) were collected and analysed with SDS/
PAGE. Peak fractions containing SNLRP were pooled and
stored at )20 °C until use.
Protein sequencing
Purified recombinant proteins were analysed by SDS/
PAGE and transferred to ProBlott
TM
membranes (Applied

Biosystems, Foster City, CA, USA) using a Trans-blot SD
semidry transfer cell. Proteins were excised from the blots
and sequenced on a Procise 491 cLC protein sequencer
(Applied Biosystems).
Enzyme assay
The N-glycosylase activity of crude tobacco extracts or
recombinant proteins towards rRNA was determined as
described previously using rabbit reticulocyte ribosomes as
substrate [32].
The PAG activity of the native RIPs was determined by
measuring the adenine released from the substrate as
described by Brigotti et al. [33]. Reactions were performed
in Eppendorf tubes containing 1 pmol RIP and 5 lg
substrate (TMV genomic RNA) in 50 lL PAG buffer
(50 m
M
sodium acetate, 100 m
M
KCl, pH 4.0). After
40 min incubation at 30 °C, the adenine released was
quantified by LC/MS on a Waters Alliance/ZQ apparatus
(Waters Corporation, Milford, MA, USA).
TMV bioassays
Based on the molecular analysis, five transgenic lines per
construct (SNA-I, SNA-IV, SNA-V, SNLRP) were ran-
domly chosen to study their level of protection against TMV
infection. Wild-type and transgenic (T
1
-generation) tobacco
plants were grown to the six-leaf stage in a culture room at

22 °C under a 16 h photoperiod. Plants were mechanically
inoculated by rubbing the upper two fully expanded leaves
with a virus suspension [1 lgÆmL
)1
TMV in 0.1
M
KH
2
PO
4
,
2% (w/v) polyvinylpyrrolidone, pH 7.2] in the presence of
carborundum. After infection, plants were maintained in
a greenhouse at 22 °C, 50% relative humidity, and a 16 h
photoperiod. Four days after infection the total number of
lesions on both leaves was counted. To minimize variation,
a complete randomised block design was used. Experiments
were repeated three times. Before analysis, the dataset was
transformed to the logarithmic scale. The transformed data
1510 F. Vandenbussche et al.(Eur. J. Biochem. 271) Ó FEBS 2004
were analysed using analysis of variance, and means were
compared using a Tukey’s HSD multiple range test
(a ¼ 0.05).
Induction of PR proteins
The possible constitutive expression of PR proteins in
transgenic plants expressing the different RIPs/lectins was
checked by Northern blot analysis. Total RNA was
isolated, separated by agarose gel electrophoresis, and
blotted. Blots were hybridized with specific probes for PR-1,
PR-2, PR-3 and proteinase inhibitor II [34].

Results and discussion
PAG activity towards TMV genomic RNA
To corroborate the possible relation between the PAG and
antiviral activity of RIPs, native elderberry RIPs were tested
for their depurinating activity towards TMV genomic RNA
(Table 1). Of the RIPs tested, both SNA-V and SNLRP
exhibited weak PAG activity towards TMV genomic RNA,
whereas SNA-I showed no activity under the same experi-
mental conditions. This complete lack of PAG activity for
SNA-I is most probably due to its intrinsically lower
enzymatic activity. As previously shown, SNA-I exhibits a
markedly lower PAG activity on different nucleic acid
substrates than other type-2 RIPs (possibly as a result of its
complex tetrameric structure) [35,36]. As none of the
elderberry RIPs display enzymatic activity towards plant
ribosomes in rRNA N-glycosylase activity assays [9], they
are ideal candidates for assessing whether the PAG activity
is involved in RIP-mediated protection.
Expression of SNA-I, SNA-IV, SNA-V and SNLRP
in transgenic tobacco
Tobacco leaf discs were transformed with A. tumefaciens
GV3101 harboring the various pGKrip/lectin vectors. The
RIP/lectin-expressing plants are hereafter referred to as
RIP/n where ÔRIPÕ denotes the RIP/lectin expressed and ÔnÕ
the number. No ÔvisibleÕ phenotypic aberrations were
observed in any of the RIP/lectin-expressing transformants,
indicating that these elderberry type-2 RIPs exert no
cytotoxic effects in planta.
All plants obtained after transformation were analysed by
PCR to confirm the presence of the RIP/lectin coding

sequence in the tobacco genome. Only those plants that
were positive in the PCR analysis were withheld for further
analysis. Expression of the RIP/lectin transgenes was
examined by analysing all transgenic lines (T
0
generation)
at the RNA and protein level. Transcription products of the
predicted size were detected in most transformants but
never in wild-type plants (data not shown). Western blot
analysis of crude leaf extracts confirmed that most of these
transformants contained immunoreactive bands. As only a
limited number of transformants could be analysed in the
viral bioassay, five lines per construct were randomly chosen
for a more detailed analysis. To estimate the expression
levels of the RIPs/lectins, transgenic lines (T
1
generation)
were analysed for rRIP/lectin content by Western blot
densitometry (Fig. 1, Table 2). The highest expression levels
were observed for the SNA-V lines, in which the RIP
accounted for 0.8–5.0% of the total soluble leaf protein. All
other lines exhibited markedly lower expression levels of
recombinant RIPs/lectins, varying between 0.03% and
1.7% of the total soluble protein. To verify whether the
rRIPs are enzymatically active, crude extracts from four
different RIP/lectin-expressing transformants (SNA-I/4,
SNA-IV/3, SNA-V/4 and SNLRP/2) were analysed for
the presence of rRNA N-glycosylase activity. The SNA-IV
transformant was included as a negative control. As shown
in Fig. 2A, the characteristic Endo fragment was only

detected in the SNA-V and SNLRP transformants (lanes 6
and 8), but not in the SNA-I and SNA-IV transformants
(lanes 2 and 4). The lack of enzymatic activity in the SNA-I
Table 1. RIP-catalysed release of adenine (pmol per pmol RIP per
40 min) from TMV genomic RNA.
RIP Adenine released
SNA-I 0.0 – trace
SNA-V 0.45 ± 0.12
SNLRP 0.57 ± 0.16
a
a
Statistically significantly at P<0.02.
Fig. 1. Western blot analysis of total soluble protein from wild-type and
transgenic tobacco plants. Approximately 15 lg (SNA-I, SNA-IV),
7.5 lg (SNLRP) or 2.5 lg (SNA-V) total soluble protein was separ-
ated in a 15% (w/v) SDS/polyacrylamide gel and transferred to a nylon
membrane. Membranes were probed with polyclonal rabbit antibodies
raised against the different RIPs/lectins. Sample loading was as fol-
lows:lane–,wild-type;lane+,purenativeRIP/lectin;lanes1–5,RIP/
1–5; lane 6–12, serial dilution (400–6.25 ng) of native pure RIP/lectin.
Table 2. Expression levels (% of total soluble protein) of recombinant
elderberry RIPs/lectins in transgenic tobacco plants in the different line
numbers.
RIP 1 2345
SNA-I 0.2 0.4 0.4 0.4 0.4
SNA-IV 0.03 1.0 1.7 0.4 0.4
SNA-V 4.3 0.8 3.7 5.0 2.4
SNLRP 0.3 0.8 0.1 0.8 0.6
Ó FEBS 2004 Antiviral activity of ribosome-inactivating proteins (Eur. J. Biochem. 271) 1511
transformants was presumably due to the lower expression

levels in these transformants and the intrinsically lower
enzymatic activity of SNA-I.
Purification and characterization of recombinant proteins
Recombinant SNA-I, SNA-IV, SNA-V and SNLRP were
purified from leaves of transgenic tobacco plants, and their
purity confirmed by SDS/PAGE and Western blot analysis.
Staining of the gels with Coomassie Brilliant Blue yielded
virtually identical migration patterns for the native and
recombinant proteins (Fig. 3), indicating that the rRIPs/
lectins were electrophoretically pure. Moreover, the pres-
ence of high-molecular-mass bands in the lanes with
unreduced rSNA-I suggests that it adopts the same
[A-s-s-B-s-s-B-s-s-A]
2
structure as native SNA-I. However,
the presence of a faster migrating band in unreduced rSNA-
I suggests that the intermolecular disulfide bridge formation
between the two [A-s-s-B] pairs is less efficient in tobacco
than in elderberry. Upon reduction, the high-molecular-
mass bands of both rSNA-I and SNA-I disappeared giving
rise to two polypeptides of nearly identical mass. All other
RIPs (SNA-V, SNLRP) yielded a banding pattern typical
of type-2 RIPs (i.e. characterized by the presence of two
distinct polypeptides of  30 and 35 kDa, respectively),
indicating that they are composed of [A-s-s-B] protomers.
As antibodies to SNA-II were used to detect SNA-V, no
antibodies directed against the A chain were present and
accordingly only the B chain of SNA-V could be visualized
in the Western blot analysis. In contrast with elderberry
bark where  95% of the SNA-V precursor is converted

into SNA-II and only 5% in SNA-V, the SNA-V-expressing
tobacco plants contained little or no rSNA-II, indicating
that in tobacco the precursor of rSNA-V is exclusively
converted into the genuine type-2 RIP SNA-V. The
molecular structure of the recombinant RIPs/lectins was
further analysed by gel filtration on a Superose 12 column.
As expected, the native and recombinant proteins were
eluted at the same position (data not shown). Furthermore
N-terminal sequencing of the recombinant proteins con-
firmed that tobacco cells successfully recognize and cleave
the signal peptide as well as the internal linker peptide at
exactly the same positions as in the parent plant (data not
shown). Analysis of the rRNA N-glycosylase activity using
rabbit ribosomes as a substrate demonstrated that both
purified rSNA-V and rSNLRP exhibited strong enzymatic
activity (Fig. 2B, lanes 6 and 8) whereas no activity could be
detected for the purified rSNA-IV (lane 4), which served as a
negative control. However, in contrast with the results of the
enzymatic assays with crude extracts, a clear signal was also
observed for purified rSNA-I (lanes 2). These results clearly
show that all rRIPs are enzymatically active. Similar results
wererecentlyalsoreportedforSNA-IfandSNA-I¢ [37,38].
Fig. 2. rRNA N-glycosylase activity of crude extracts from five different
RIP/lectin-expressing transformants (A) or purified recombinant RIPs/
lectins (B) towards rabbit reticulocyte ribosomes. The arrow indicates
the position of the Endo fragment released from the rRNA. (–) and
(+) indicate no treatment and aniline treatment, respectively. Sample
loading was as follows: lanes 1 and 2, SNA-I/4; lanes 3 and 4, SNA-IV/
3; lanes 5 and 6, SNA-V/4; lanes 7 and 8, SNLRP/2.
Fig. 3. SDS/PAGE (left hand panel) and Western blot (right hand

panel) analysis of purified native and recombinant RIPs/lectins under
nonreducing (A) and reducing (B) conditions. Sample loading was as
follows:lane1,SNA-I;lane2,rSNA-I;lane3,SNA-IV;lane4,rSNA-
IV;lane5,SNA-V;lane6,rSNA-V;lane7,SNLRP;lane8,rSNLRP.
Molecular mass markers were phosphorylase b (97 kDa), albumin
(66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin
inhibitor (20 kDa) and lysozyme (14 kDa).
1512 F. Vandenbussche et al.(Eur. J. Biochem. 271) Ó FEBS 2004
The results presented here and elsewhere thus indicate that
tobacco is not only capable of correctly processing and
assembling monomeric (SNLRP) but also dimeric (SNA-I¢,
SNA-V) and even tetrameric (SNA-I, SNA-If) type-2 RIPs.
TMV bioassays
To assess whether and, if so, to what extent the elderberry
RIPs provide in planta protection against viruses (non)trans-
genic control plants and RIP-expressing tobacco plants (T
1
generation) were challenged with TMV. For each RIP, five
transgenic lines were randomly chosen to be analysed in the
bioassays. As SNA-IV lacks the catalytically active A chain,
it is expected to confer no protection against viruses and
hence serves as a negative control. To minimize variation, a
complete randomised block design was used, with each
block consisting of a nontransgenic control plant (wild-
type), a transgenic control plant (empty-vector) and five
RIP-expressing transgenic plants (RIP/1–5). Three inde-
pendent experiments were set up for each RIP. Experimen-
tal data were combined to calculate the mean number of
lesions, which were compared using a Tukey’s HSD
multiple range test (a ¼ 0.05). Significant differences

between control and RIP-expressing plants were only
observed for SNA-V (SNA-V/1, SNA-V/3, SNA-V/4)
(Table 3). Interestingly, a direct correlation was observed
between the expression level of rSNA-V and the level of
protection against TMV in the different SNA-V lines. Only
the three lines with the highest protection levels showed a
significant reduction in lesion numbers compared with
control plants. Although these results show that the type-2
RIP SNA-V is capable of conferring local protection against
TMV infection, the level of protection is markedly lower
than that of type-1 RIPs. On average, the three highest
expressing SNA-V lines showed a reduction in lesion
numbers of 39%. Using the same bioassay, Desmyter et al.
[31] showed that IRIP, a type-1 RIP from Iris hollandica L.,
provides high levels of protection for transgenic tobacco
plants, with an average reduction in lesion numbers of 75%.
Moreover, all five IRIP-expressing lines exhibited similar
protection levels. The observation that SNA-V provides
protection only at very high expression levels (> 3.7% of the
total soluble protein) suggests that the antiviral mechanism
of SNA-V differs from that of IRIP. Like most type-1 RIPs,
IRIP exhibits rRNA N-glycosylase activity towards both
animal and plant ribosomes [39], and accordingly is capable
of depurinating in planta part of the ribosomes in IRIP-
expressing tobacco on TMV infection (as could be demon-
strated by an in planta depurination assay) [40]. Thus, the
strong in planta antiviral activity of IRIP is presumably due
to its direct depurinating activity towards both viral nucleic
acids and plant ribosomes and hence relies on a direct effect
on both the invading viruses and the infected cells. As SNA-

V does not act on plant ribosomes, the elderberry RIP can
only act on the virus itself and not on the infected cells, which
may explain why it provides less protection against viral
infection than IRIP or other type-1 RIP.
Although both SNA-V and SNLRP exhibited similar
depurination rates on TMV genomic RNA (Table 1), only
the highest SNA-V-expressing lines showed a significant
reduction in TMV lesion numbers. However, as mentioned
above, the SNA-V lines showed markedly higher expression
levels than any of the other RIP-expressing tobacco lines.
These results suggest that in planta depurination of viral
nucleic acids requires high cellular RIP concentrations.
Similar results were recently reported for the cap-specific
depurination by the type-1 RIP PAP from pokeweed [41].
Using a filter-binding assay, the authors demonstrated that
PAP has a nearly fourfold lower affinity for capped RNAs
than for rRNA. As a consequence, PAP is only expected to
significantly interact with capped RNAs at high cellular
concentrations and/or high capped RNA levels. It is likely
therefore that the ectopically expressed SNLRP exerts no
protection in planta because its expression level remains
below the threshold concentration required for activity.
Analysis of PR protein expression in transgenic
tobacco plants
As according to previous reports the ectopic expression of
PAP and various PAP mutants resulted in constitutive
expression of some PR proteins [42,43], it seemed worth
Table 3. Susceptibility of wild-type and transgenic tobacco plants to
TMV infection. The mean number of lesions was calculated for each
RIP separately and compared using a Tukey’s HSD multiple range test

(a ¼ 0.05). Inhibition percentages were calculated by the following
formula: 100 · (number of lesions wild-type – number of lesions
transformant)/number of lesions wild-type. When the number of
lesions of the transformant was as high or higher than the wild-type
value, the inhibition percentage was set to zero.
Tobacco line
Number
of plants
Number of
lesions
Inhibition
(%)
Wild-type 26 151 ± 67 A 0
Empty vector 26 140 ± 47 A 7
SNA-I/1 26 113 ± 56 A 25
SNA-I/2 25 133 ± 68 A 12
SNA-I/3 26 140 ± 69 A 7
SNA-I/4 26 122 ± 57 A 19
SNA-I/5 25 159 ± 63 A 0
Wild-type 24 149 ± 60 A 0
Empty vector 24 152 ± 27 A 0
SNA-IV/1 24 158 ± 56 A 0
SNA-IV/2 24 145 ± 69 A 3
SNA-IV/3 24 131 ± 63 A 12
SNA-IV/4 24 112 ± 37 A 25
SNA-IV/5 24 123 ± 36 A 17
Wild-type 28 146 ± 77 A 0
Empty vector 28 141 ± 62 A 3
SNA-V/1 28 88 ± 40 B 40
SNA-V/2 28 160 ± 81 A 0

SNA-V/3 28 83 ± 47 B 43
SNA-V/4 28 97 ± 57B 34
SNA-V/5 26 185 ± 90 A 0
Wild-type 24 208 ± 128 A 0
Empty vector 24 167 ± 99 A 20
SNLRP/1 24 196 ± 94 A 6
SNLRP/2 24 197 ± 115 A 5
SNLRP/3 24 179 ± 93 A 14
SNLRP/4 24 237 ± 98 A 0
SNLRP/5 24 198 ± 88 A 5
Ó FEBS 2004 Antiviral activity of ribosome-inactivating proteins (Eur. J. Biochem. 271) 1513
while to check whether the same phenomenon occurs in
tobacco plants that synthesize one of the elderberry type-2
RIPs/lectins. Therefore, the presence of mRNAs encoding
PR-1, PR-2, PR-3 and proteinase inhibitor II was verified
by Northern blot analysis. In none of the transgenic plants
could any of these mRNAs be detected (data not shown),
indicating that constitutive expression of neither the type-2
RIPs SNA-V, SNA-I and SNLRP nor the lectin SNA-IV
was accompanied by an increase in acidic or basic PR
proteins. Similar results were recently also reported for
transgenic tobacco plants expressing IRIP or SNA-I¢
[31,37,38]. In spite of enhanced protection levels against
TMV infection, none of these plants constitutively accu-
mulated PR proteins. The results presented here and
elsewhere thus clearly indicate that the proposed mode of
action of PAP (i.e. through activation of the plant’s defence
system) cannot be generalized to other RIPs.
Acknowledgements
This work was supported in part by grants from the Katholieke

Universiteit Leuven and DG6 Ministerie voor Middenstand en
Landbouw-Bestuur voor Onderzoek en Ontwikkeling. P. P. is a
Postdoctoral Fellow of the Fund for Scientific Research-Flanders. The
work performed in Bologna was partially supported by Progetto
Strategico Oncologia n.74 (DD 19Ric, 09/01/02) from Ministero
Istruzione Universita
`
e Ricerca, Italy. The TMV strain was a gift from
Professor M. Ho
¨
fte (Laboratory of Phytopathology, Ghent University,
Belgium).
References
1. Endo, Y., Mitsui, K., Motizuki, M. & Tsurugi, K. (1987) The
mechanism of action of ricin and related toxic lectins on eu-
karyotic ribosomes. The site and the characteristics of the mod-
ification in 28 S ribosomal RNA caused by the toxins. J. Biol.
Chem. 262, 5908–5912.
2. Endo, Y. & Tsurugi, K. (1987) RNA N-glycosidase activity of
ricin A-chain. Mechanism of action of the toxic lectin ricin on
eukaryotic ribosomes. J. Biol. Chem. 262, 8128–8130.
3. Van Damme, E.J.M., Hao, Q., Chen, Y., Barre, A., Vandenbus-
sche, F., Desmyter, S., Rouge
´
, P. & Peumans, W.J. (2001) Ribo-
some-inactivating proteins: a family of plant proteins that do more
than inactivate ribosomes. Crit. Rev. Plant Sci. 20, 395–465.
4. Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B. & Peumans,
W.J. (1984) A lectin from elder (Sambucus nigra L.) bark. Bio-
chem. J. 221, 163–169.

5. Kaku, H., Peumans, W.J. & Goldstein, I.J. (1990) Isolation
and characterization of a second lectin (SNA-II) present in el-
derberry (Sambucus nigra L.) bark. Arch. Biochem. Biophys. 277,
255–262.
6. Van Damme, E.J.M., Barre, A., Rouge
´
,P.,VanLeuven,F.&
Peumans, W.J. (1996) The NeuAc (alpha-2,6) -Gal/GalNAc-
binding lectin from elderberry (Sambucus nigra) bark, a type-2
ribosome-inactivating protein with an unusual specificity and
structure. Eur. J. Biochem. 235, 128–137.
7. Van Damme, E.J.M., Roy, S., Barre, A., Citores, L., Mostafa-
pous, K., Rouge
´
, P., Van Leuven, F., Girbes, T., Goldstein, I.J. &
Peumans, W.J. (1997) Elderberry (Sambucus nigra)barkcontains
two structurally different Neu5Ac(alpha2,6)Gal/GalNAc-binding
type 2 ribosome-inactivating proteins. Eur. J. Biochem. 245,
648–655.
8. Van Damme, E.J.M., Barre, A., Rouge
´
,P.,VanLeuven,F.&
Peumans, W.J. (1996) Characterization and molecular cloning of
Sambucus nigra agglutinin V (nigrin b), a GalNAc-specific type-2
ribosome-inactivating protein from the bark of elderberry (Sam-
bucus nigra). Eur. J. Biochem. 237, 505–513.
9. Girbes, T., Citores, L., Ferreras, J.M., Rojo, M.A., Iglesias, R.,
Munoz, R., Arias, F.J., Calonge, M., Garcia, J.R. & Mendez, E.
(1993) Isolation and partial characterization of nigrin b, a non-
toxic novel type 2 ribosome-inactivating protein from the bark of

Sambucus nigra L. Plant Mol. Biol. 22, 1181–1186.
10. Van Damme, E.J.M., Roy, S., Barre, A., Rouge
´
,P.,VanLeuven,
F. & Peumans, W.J. (1997) The major elderberry (Sambucus nigra)
fruit protein is a lectin derived from a truncated type 2 ribosome-
inactivating protein. Plant J. 12, 1251–1260.
11. Van Damme, E.J.M., Barre, A., Rouge
´
,P.,VanLeuven,F.&
Peumans, W.J. (1997) Isolation and molecular cloning of a novel
type 2 ribosome-inactivating protein with an inactive B chain from
elderberry (Sambucus nigra)bark.J. Biol. Chem. 272, 8353–8360.
12. Barbieri, L., Ferreras, J.M., Barraco, A., Ricci, P. & Stirpe, F.
(1992) Some ribosome-inactivating proteins depurinate ribosomal
RNA at multiple sites. Biochem. J. 286, 1–4.
13. Barbieri, L., Gorini, P., Valbonesi, P., Castiglioni, P. & Stirpe, F.
(1994) Unexpected activity of saporins. Nature (London) 372,624.
14. Barbieri, L., Valbonesi, P., Gorini, P., Pession, A. & Stirpe, F.
(1996) Polynucleotide:adenosine glycosidase activity of sapor-
in-L1: effect on DNA, RNA and poly (A). Biochem. J. 319, 507–
513.
15. Barbieri,L.,Valbonesi,P.,Govoni,M.,Pession,A.&Stirpe,F.
(2000) Polynucleotide:adenosine glycosidase activity of saporin-
L1: effect on various forms of mammalian DNA. Biochim.
Biophys. Acta 1480, 258–266.
16. Barbieri, L., Valbonesi, P., Bonora, E., Gorini, P., Bolognesi, A. &
Stirpe, F. (1997) Polynucleotide:adenosine glycosidase activity of
ribosome-inactivating proteins: effect on DNA, RNA and poly
(A). Nucleic Acids Res. 25, 518–522.

17. Bolognesi, A., Polito, L., Olivieri, F., Valbonesi, P., Barbieri, L.,
Battelli, M.G., Carusi, M.V., Benvenuto, E., Del Vecchio, B.F., Di
Maro, A., Parente, A., Di Loreto, M. & Stirpe, F. (1997) New
ribosome-inactivating proteins with polynucleotide:adenosine
glycosidase and antiviral activities from Basella rubra L. & Bou-
gainvillea spectabilis Willd. Planta 203, 422–429.
18. Di Maro, A., Valbonesi, P., Bolognesi, A., Stirpe, F., De Luca, P.,
Siniscalco, G.G., Gaudio, L., Delli, B.P., Ferranti, P., Malorni, A.
& Parente, A. (1999) Isolation and characterization of four type-1
ribosome-inactivating proteins, with polynucleotide:adenosine
glycosidase activity, from leaves of Phytolacca dioica L. Planta
208, 125–131.
19. Rajamohan, F., Venkatachalam, T.K., Irvin, J.D. & Uckun, F.M.
(1999) Pokeweed antiviral protein isoforms PAP-I, PAP-II, and
PAP-III depurinate RNA of human immunodeficiency virus
(HIV)-1. Biochem. Biophys. Res. Commun. 260, 453–458.
20. Bolognesi, A., Polito, L., Lubelli, C., Barbieri, L., Parente, A. &
Stirpe, F. (2002) Ribosome-inactivating and adenine polynucleo-
tide glycosylase activities in Mirabilis jalapa L. tissues. J. Biol.
Chem. 277, 13709–13716.
21. Peumans, W.J., Hao, Q. & Van Damme, E.J.M. (2001) Ribo-
some-inactivating proteins from plants: more than RNA N-gly-
cosidases? FASEB J. 15, 1493–1506.
22. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.
23. Becker, D., Kemper, E., Schell, J. & Masterson, R. (1992) New
plant binary vectors with selectable markers located proximal to
the left T-DNA border. Plant Mol. Biol. 20, 1195–1197.
24. Timmermans, M., Maliga, P., Vieira, J. & Messing, J. (1990) The

pFF plasmids: cassettes utilising CaMV sequences for expression
of foreign genes in plants. J. Biotechnol. 14, 333–334.
1514 F. Vandenbussche et al.(Eur. J. Biochem. 271) Ó FEBS 2004
25. Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing
with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74,
5463–5467.
26. Shen, W.J. & Forde, B.G. (1989) Efficient transformation of
Agrobacterium spp. by high voltage electroporation. Nucleic Acids
Res. 17, 8385.
27. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers,
S.G. & Fraley, R.T. (1985) A simple and general method for
transferring genes into plants. Science 227, 1229–1231.
28. Goode, B.L. & Feinstein, S.C. (1992) ÔSpeedprepÕ purification of
template for double-stranded DNA sequencing. Biotechniques 12,
374–375.
29. Eggermont, K., Goderis, I.J. & Broekaert, W.F. (1996) High-
throughput RNA extraction from plant samples based on
homogenisation by reciprocal shaking in the presence of a mixture
of sand and glass beads. Plant Mol. Biol. Rep. 14, 273–279.
30. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
31. Desmyter, S., Vandenbussche, F., Hao, Q., Proost, P., Peumans,
W.J. & Van Damme, E.J.M. (2003) Type-1 ribosome-inactivating
protein from iris bulbs: a useful agronomic tool to engineer virus
resistance? Plant Mol. Biol. 51, 567–576.
32. Van Damme, E.J.M., Hao, Q., Charels, D., Barre, A., Rouge
´
,P.,
Van Leuven, F. & Peumans, W.J. (2000) Characterization and

molecular cloning of two different type 2 ribosome-inactivating
proteins from the monocotyledonous plant Polygonatum multi-
florum. Eur. J. Biochem. 267, 2746–2759.
33. Brigotti, M., Alfieri, R., Sestili, P., Bonelli, M., Petronini, P.G.,
Guidarelli,A.,Barbieri,L.,Stirpe,F.&Sperti,S.(2002)Damage
to nuclear DNA induced by Shiga toxin 1 and ricin in human
endothelial cells. FASEB J. 16, 365–372.
34. Ward, E.R., Uknes, S.J., Williams, S.C., Dincher, S.S., Wieder-
hold, D.L., Alexander, D.C., Ahl-Goy, P., Me
´
traux, J P. & Ryals,
J.A. (1991) Coordinate gene activity in response to agents that
induce systemic acquired resistance. Plant Cell 3, 1085–1094.
35. Battelli, M.G., Barbieri, L., Bolognesi, A., Buonamici, L.,
Valbonesi, P., Polito, L., Van Damme, E.J.M., Peumans, W.J. &
Stirpe, F. (1997) Ribosome-inactivating lectins with poly-
nucleotide:adenosine glycosidase activity. FEBS Lett. 408,
355–359.
36. Barbieri, L., Ciani, M., Girbes, T., Liu, W Y., Van Damme,
E.J.M., Peumans, W.J. & Stirpe, F. (2004) Enzymatic activity of
toxic and non-toxic type 2 ribosome-inactivating proteins. FEBS
Lett. in press.
37. Chen, Y., Vandenbussche, F., Rouge
´
,P.,Proost,P.,Peumans,
W.J. & Van Damme, E.J.M. (2002) A complex fruit-specific type-2
ribosome-inactivating protein from elderberry (Sambucus nigra)is
correctly processed and assembled in transgenic tobacco plants.
Eur. J. Biochem. 269, 2897–2906.
38. Chen, Y., Peumans, W.J. & Van Damme, E.J.M. (2002) The

Sambucus nigra type-2 ribosome-inactivating protein SNA-I¢
exhibits in planta antiviral activity in transgenic tobacco. FEBS
Lett. 516, 27–30.
39. Hao,Q.,VanDamme,E.J.M.,Hause,B.,Barre,A.,Chen,Y.,
Rouge
´
, P. & Peumans, W.J. (2001) Iris bulbs express type 1 and
type 2 ribosome-inactivating proteins with unusual properties.
Plant Physiol. 125, 866–876.
40. Desmyter, S. (2002) Study of the antiviral activity of IRIP, a type-
1 ribosome-inactivating protein from Iris hollandica.PhDThesis,
Katholieke Universiteit Leuven, Belgium.
41. Hudak, K.A., Bauman, J.D. & Tumer, N.E. (2002) Pokeweed
antiviral protein binds to the cap structure of eukaryotic mRNA
and depurinates the mRNA downstream of the cap. RNA 8,
1148–1159.
42. Zoubenko, O., Uckun, F., Hur, Y., Chet, I. & Tumer, N. (1997)
Plant resistance to fungal infection induced by nontoxic pokeweed
antiviral protein mutants. Nat. Biotechnol. 15, 992–996.
43. Zoubenko, O., Hudak, K. & Tumer, N.E. (2000) A non-toxic
pokeweed antiviral protein mutant inhibits pathogen infection via
a novel salicylic acid-independent pathway. Plant Mol. Biol. 44,
219–229.
Ó FEBS 2004 Antiviral activity of ribosome-inactivating proteins (Eur. J. Biochem. 271) 1515