Tài liệu Báo cáo khoa học: Specific interaction between the classical swine fever virus NS5B protein and the viral genome pdf

Specific interaction between the classical swine fever virus NS5B
protein and the viral genome
Ming Xiao
1,2
, Jufang Gao
2
, Wei Wang
2
, Yujing Wang
2
, Jun Chen
2
, Jiakuan Chen
1
and Bo Li
1
1
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, The Institute of Biodiversity Science,
Fudan University, Shanghai, China;
2
College of Life and Environment Sciences, Shanghai Normal University, China
The NS5B protein of the classical sw ine fever virus ( CSFV) is
the RNA-dependent RNA polymerase of the virus and is
able to catalyze the viral genome replication. The 3¢ un-
translated region is most likely involved in regulation of t he
Pestivirus genome replication. However, little is known
about the i nteraction between the CSFV NS5B p rotein and
the viral genome. We used different RNA templates derived
from the plus-strand viral genome, or the minus-strand viral
genome and the C SFV NS 5B protein obtained f rom t he
Escherichia coli expression system to address th is problem.

We first showed that the viral NS5B protein formed a
complex with the plus-strand g enome t hrough t he genomic
3¢ UTR and that the NS5B protein was also able to bind the
minus-strand 3¢ UTR. Moreover, it was found that viral
NS5B protein bound the minus-strand 3 ¢ UTR more effi-
ciently than the plus-strand 3¢ UTR. Furth er, we obse rved
that the plus-strand 3¢ UTR with deletion of CCCGG or 21
continuous nucleotides a t its 3¢ terminal had no b inding
activity and also l ost the activity for i nitiation of m inus-
strand RNA synthesis, which similarly occurred in the
minus-strand 3¢ UTR with CATATGCTC or the 21 nuc-
leotide f ragment deleted from the 3¢ terminal. Therefore, it is
indicated that the 3¢ CCCGG sequence of the plus-strand
3¢ UTR, and t he 3¢ CATATGCTC f ragment o f the minus-
strand are essential to in vitro syn thesis o f t he minus-strand
RNA a nd the plus-strand RNA, respectively. The same
conclusion is also appropriate for the 3¢ 21 nucleotide
terminal site of both the 3¢ UTRs.
Keywords: CSFV; RdRp; replication; RNA synthesis;
3¢ UTR.
Classical swine fever virus (CSFV) is the causative agent of
swine fever, which is a h ighly contagious and f atal viral
disease o f pigs. CSFV, bovine v iral diarrhea virus (BVDV),
and Border Disease virus (BDV) are members of the
Pestivirus genus within the Flaviviridae family. BVDV a nd
BDV can infect both ruminants and pigs. The h epatitis C
virus (HCV), an etiological agent of non-A, non-B hepatitis,
also belongs to the Flaviviridae family.
Pestiviruses are small, enveloped, plus-strand RNA
viruses, similar t o HCV. The RNA genome is  12.5 kb in

length, consisting of a large and continual open r eading
frame (ORF), a 5¢ untranslated region (5¢ UTR) and a 3¢
untranslated region (3¢ UTR). The ORF is translated into a
polyprotein, which is further processed into 12 mature
proteins b y viral and host cell proteases. The 12 proteins
comprise four structure proteins (C, E
rns
, E1, and E2) and
eight nonstructure proteins (N
pro
, P7, NS2, NS3, NS4A,
NS4B, NS5A, and NS5B). In the CSFV genome, the genes
encoding N
pro
,C,E
rns
, E1, E2, p7, and NS2 have proved to
be dispensable for RNA replication [1]. The 3¢ UT R and the
5¢ UTRarebelievedtoregulatePestivirus genome replica-
tion [2,3]. The Pestivirus genomic replication c onsists of
several c onsecutive processes. Repliase first recognizes and
binds the 3¢ UTRandstartsRNAsynthesis,inwhicha
minus-strand RNA is produced with the plus-strand
genomic RNA as a template. Then, a progeny plus-RNA
is produced with the novel minus-RNA as a template [4]. The
5¢ UTR is also the site fo r initiating t ranslation of the v iral
genomes, at which an internal ribosomal entry site (IRES) is
observed [5]. Short 3¢ terminal extensions do not interfere
with infectivity of in vitro transcript whereas 5¢ extensions
sometimes do and sometimes do not [6]. The CSFV NS5B

gene is located a t the 3¢ end of the genome adjacent to the
3¢ UTR. The CSFV NS5B protein has an RNA-dependent
RNA polymerase (RdRp) activity, and thus plays a central
role in viral RNA replication [7–10]. Even NS5B as a fusion
protein with the green fluorescent p rotein still displays an
RdRp activity [11]. The NS5B proteins of BVDV and HCV
have been expressed in different systems and their biochemi-
cal properties h ave been s tudied [12–17]. N S5B protein is
able to catalyze RNA elongation b y a primer-dependent o r
copy-back mechanism, and can initiate RNA synthesis from
the 3¢ end of different RNA templates in vitro [7,9,15]. I t is
reported t hat t he mechanism for de novo initiation of RNA
synthesis is a lso a ssociated with t he NS5B proteins [17–21].
Moreover, the crystal structure of HCV NS5B protein has
been characterized [22].
Correspondence to B. Li, Ministry of Education Key Laboratory for
Biodiversity Science and Ecological Engineering, The Institute of
Biodiversity Science, Fudan University, Shanghai, 200433, China.
Fax: +86 21 6564246, Tel.: +86 2 1 65642178,
E-mail:
Abbreviations: BDV, Border disease virus; BVDV, bovine viral
diarrhea virus; CSFV, classical swine fever virus; EMSA, electro-
phoretic mobility shift assay; HCV, hepatitis C virus; IRES, internal
ribosome entry site; RdRp, RNA-dependent RNA polymerase;
TNTase, terminal nucleotidyl transferase.
(Received 25 June 2004, revised 28 July 2004, accepted 6 August 2004)
Eur. J. Biochem. 271, 3888–3896 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04325.x
Studies of pestiviral replication have been hampered b y
the lack of efficient culture systems. Most of the informa-
tion on cis-acting sequences of these v iral genomes comes

from the research o n H CV [23 –26]. Recently, the studies of
the cis-acting sequences of the CSFV genome have been
performed in vitro. A 21 nucleotide fragment at the 3¢
terminal of the 3¢ UTR m ay be essential to initiation of
RNA s ynthesis [7,27]. Furthermore, i t i s found that the
12 nucleotide insertion ÔCTTTTTTCTTTTÕ present in the
3¢ UTR of the CSFV HCLV strain [28], an efficient
vaccine strain, might be responsible for a virulence [29]. But,
the binding of the CSFV NS5B t o the cis-acting sequences
relative to the viral replication has not yet been character-
ized. In this study, we have performed competitive
electrophoretic mobility shift assays (EMSA) and R dRp
assays, and examined the influence of d ifferent cis-acting
elements on binding of NS5B protein and RNA synthesis
in vitro.
Materials and methods
Expression and purification of NS5B proteins
The recombinant plasmid for expression of CSFV NS5B
protein was constructed as described pr eviously [7]. Total
RNA was extracted from CSFV Shimen s train. A f ull-
length NS5B cDNA was obtained by RT-PCR, and cloned
into the pET28(a) vectors. A methionine codon for
initiating translation was added to the 5¢ end of the NS5B
coding sequence. Additional sequences coding for six
histidines at the C-terminus were engineered to facilitate
the purification of NS5B protein. The i nserted regions of all
clones were sequenced through d ideoxynucleotide sequen-
cing and no c hanges were found. These resulting plasmids
were introduced into Escherichia c oli strain BL21DE3 for
expression driven by the bacteriophage T7 RNA poly-

merase. T he E. coli strain BL21DE3 cells were cultured in
M9ZB media with 40 lgÆmL
)1
kanamycin at 37 °C.
Expression was induced by addition of iso propyl thio-
b-
D
-galactoside. Extraction and purification were per-
formed as described previously [17]. Briefly, the bacterial
cell culture was harvested and washed w ith phosphate-
buffered saline ( NaCl/P
i
). The cells from 1000 mL were
resuspended in 20 mL of the buffer containing 50 m
M
Na-phosphate (pH 8.0), 300 m
M
NaCl, 10 m
M
imidazole,
10 m
M
2-mercaptoethano l, 10% ( v/v) glycerol, 1 % (v/v)
Nonidet P-40, supplemented with 1 m
M
phenylmethylsulfo-
nyl fluoride and 10 m
M
leupeptin. After undergoing
freezing and thawing once, c ells were subjected to sonica-

tion. The cleared lysate was obtained by centrifugation at
35 000 g for 15 min. The cleared lysate containing the
recombinant protein was purified using Ni-nitrilotriacetic
acid–Sepharose resin ( Gibco BRL). Briefly, t he CSFV
NS5B with a polyhistidine tag was bound to the
Ni-nitrilotriacetic acid resin pre-equilibrated w ith the above
buffer, and then w ashed with buffer containing 50 m
M
imidazole. The bound NS5B was eluted with buffer
containing different c oncentrations of imidazole (100 m
M
to 500 m
M
). The N S5B protein was c ollected, combined
and dialyzed in buffer A [50 m
M
Tris/HCl (pH 8.0), 1 m
M
dithiothreitol, 50 m
M
NaCl, 5 m
M
MgCl, 10% (v/v)
glycerol]. NS5B proteins were quantified as described
previously [15]. In brief, NS5B protein solutions and
dilutions of bovine serum albumin with known concentra-
tion were subjected to SDS/PAGE. The gels with the
samples were staine d with Coomassie B rilliant Blue. T he
amount of NS5B protein was determined by densitometric
scanning and comparing the two samples on the same gel.

Purified proteins were separated by SDS/PAGE, and
immunoblotted with a nti-His6 tag monoclonal antibody.
RNA preparation
The RNAs were generated as describe d previously [7]. In
brief, cDNA fragments containing complete CSFV
3¢ UTR, 5¢ UTR and random coding sequences were
initially cloned into the pGEM-T vector (Promega) f rom
total C SFV RNA by RT-PCR. These nucleotide sequences
were verified. A pair o f primers were designed on either side
of the expected mutant fragment, or the desired wild-type
sequence. The standard PCR m ethod based on the primers
was u sed. The P CR product was obtained, treated with
E. coli Klenow fragmen t, t hen with T4 DNA ligase, cloned
into the pGEM-T vector and transformed into E. coli BL21
(DE3) cells. P lasmids w ere e xtracted and sequenced. The
plasmids containing expected mutations were verified b y
sequencing. Wild-type and m utant RNA templates were
synthesized by PCR and subsequent in vitro transcription
based on these RT-PCR products. A DNA Vent poly-
merase and the primer containing bacteriophage T7
promoter were used in the PCR. After the sequence was
verified, the resulting PCR products were used as the
template for the subsequent in vitro transcription. The
in vitro transcription was performed in 50 lL o f reaction
mixtures following the standard method: 20 lLof5· tran-
scription buffer, 2 lL of RNasin (20–40 UÆmL) (Promega),
5 lL of each dNTP ( 2.5 m
M
), 5 lgofthetemplate,2lLof
T7 R NA polymerase ( 10–20 U ÆmL) (Promega). T he mix-

ture was incu bated at 37 °Cfor2h.DNaseI(10lL;
Takara) was added to the mixture and incu bated at 37 °C
for 15 min. The mixture w as extracted with phenol/chloro-
form. After ethanol precipitation, the RNA was d ried, and
redissolved in 20 lL of double distilled H
2
O. Labeled RNA
fragments were produced in the analogous way with
[
32
P]UTP[aP]. Integrity of the RNA was analyzed by
denaturing formaldehyde-agarose gel electrophoresis. The
concentration of R NA was determined b y measuring its
absorbance at 260 nm.
Competitive electrophoretic mobility shift assays
The [
32
P]UTP[aP]-labeled RNA fragment containing the
3¢ UTR of plus-strand or minus-strand genome was used as
the probe for the competitive electrophoretic mobility shift
assays ( EMSA). In e ach assay, unless otherwise specified,
1 p mol of labeled RNA was incubated w ith 400 ng of
CSFV NS5B protein in a buffer containing 20 m
M
HEPES
(pH 7.3), 5 m
M
MgCl
2
,7.5m

M
dithiothreitol, 5% (v/v)
glycerol, 125 m
M
NaCl, 100 lgÆmL
)1
bovine s erum albu-
min, 1 U of RNasin, and various amount of competitor
RNA. The reactions were performed at room temperature
for 3 0 m in. The reaction products were analyzed on a native
6% polyacrylamide g el. T he gel was dried a nd subjected to
autoradiography.
Ó FEBS 2004 Interaction between CSFV NS5B and the genome (Eur. J. Biochem. 271) 3889
RdRp assays
Total volume f or RNA polymerization t o determine the
activity of RNAs or RdRp was 50 lL, containing the
following supplements: 50 m
M
HEPES (pH 8.0), 5 m
M
MgCl, 10 l
M
dithiothreitol, 25 m
M
KCl, l m M E DTA,
20 U RNasin, 50 lg actinomycin D ( Sigma), 200 l
M
each
dNTP (including a single radiolabeled CTP, [
32

P]CTP[aP]),
1 lL of RNA template (250 ngÆmL
)1
)and50n
M
NS5B
proteins. The mixture was incubated at 37 °C for 2 h , and
the reaction was stopped by addition of 2 lLofEDTA
(200 m
M
). The reaction samples were extracted with ph enol/
chloroform, a nd RNAs were precipitated with i sopropyl
alcohol. Precipitates w ere resolved i n 2 5 lL of g el buffer
[40 m
M
MOPS (pH 7.0), 10 m
M
sodium acetate, 1 m
M
EDTA, 50% (v/v) formamide, 2.2
M
formaldehyde], heated
to 55 °C f or 15 min, then chilled o n i ce with addition of
1 lL o f e thidium b romide (10 mgÆmL
)1
). Af ter a 10 min
incubation at room temperature, 5 lL o f loading buffer
[50% (v/v) glycerol, 0.25% (v/v) bromphenol blue, 0.25%
(v/v) xylenecyanol, 1 m
M

EDTA] w as added, and samples
were loaded onto 1.5% agarose gel containing 2.2
M
formaldehyde, 40 m
M
MOPS (pH 7.0), 10 m
M
sodium
acetate, and 1 m
M
EDTA. Electrophoresis was performed
at 5 VÆcm
)1
.
Terminal nucleotidyl transferase activity assays
Terminal n ucleotidyl t ransferase activity was determined in
the same way as the RdRp assay. In brief, 10 l
M
cold UTP,
GTP, CTP, or ATP mixed with 10 lCi of the ir a
32
P-labeled
equivalent, was u sed a s the single ribonucleotide triphos-
phate in the p resence of the CSFV native 3 ¢ UTR, as a
template. T he reaction products were separated o n a 7
M
urea/20% polyacrylamide gel. At the same time, the control
experiments containing fractions isolated from untrans-
formed E. coli lysate were carried out in the same way as
above.

Results
The RdRp activity of the full-length CSFV NS5B protein
To evaluate the biological activity of CSFV RNA poly-
merase, a full-length NS5B cDNA was obtained. The
cDNA was cloned into t he pET28(a) vector, which allowed
expression of the full-length NS5B protein with a polyhis-
tidine tag in E. coli. F ollowing induction with isopropyl
thio-b-
D
-galactoside, e xtraction of bacterial lysate was
performed w ith the lysis buffe rs containing high concentra-
tions of salt, g lycerol and detergent. P urification was
preformed with N i-nitrilotriacetic acid resin. A protein with
the molecular mass equivalent to t he full-len gth CSFV
NS5B was detected in the expression product, but was not
present in a n e quivalent fraction obtained from the control
experiment. The protein was able to be immunoblotted with
anti-His6 tag monoclonal antibody, which was not found in
the control e xperiment (Fig. 1A,B). Taken together, these
results indicated that the expression product was the
recombinant NS5B protein of CSFV. When this protein
was i ncubated with the R NA templates containing the full-
length 3¢ UTR of plus-strand or minus-strand CSFV
genome ( a 6 03 or 701 nucleotide fragment, respectively)
under the conditions for RNA polymerization, newly
synthesized RNA products were detected, indicating that
the f ull-length NS5B protein was able to catalyze RNA
123
12 3
45678

UACGUACG
AC D
B
M12
50
80
97
600600
Fig. 1. Expression and RdRp-TNTase assays of the full-length CSFV N S5B protein. ( A) The N S5B proteins o btained th ro ughout expression and
extraction and purification were analyzed on SDS/PAGE and revealed by C oomassie staining. (B ) The pro teins were immunoblotted with anti-His
6
tag monoclonal antibody. Lane 1: the full-length NS5B protein. Lane 2: E. coli lysate as control. Lane M: Molecular mass markers. (C) The
products from RdRp assays at 37 °C for 2 h containing the native 3¢ UTR of the plus-strand (lane 1) or minus-strand (lane 2) RNA genome were
loaded onto 1.5% agarose gel containing 2.2
M
formaldehyde, 40 m
M
MOPS (pH 7.0), 10 m
M
sodium acetate, and 1 m
M
EDTA. Lane 3 was for
no expre ssion p rodu ct. N um bers to the left re fer t o the position of RNA c ontain ing 600 nucleotides. (D) TN Tase ac tivity assays of the N S5B
protein were condu cted with a single ribonucleotide triphosphate mixed with its equivalent labeled with [a
32
P] as described i n Materials and
methods. The r esults were shown o n a 7
M
urea/20% polyacrylamide gel.
3890 M. Xiao et al.(Eur. J. Biochem. 271) Ó FEBS 2004

synthesis with the native 3¢ UTR of the plus-strand or
minus-strand RNA genome as templates (Fig. 1C). There-
fore, t he recombinant C SFV NS5B protein expressed in
E. coli had a RNA-dependen t RNA polymerase activity.
The above experiments demonstrate that RdRp activity is
associated with the E. coli-expressed CSFV NS5B protein.
RNA f ragments longer than the templates were found in
RNA synthesis by the N S5B protein (Fig. 1C) [30]. In
addition to the R dRp activity, a terminal nucleotidyl
transferase (TNTase) function could also contribute to the
formation of these products. A TNTase could add non-
templated terminal nucleotides to either the newly synthes-
ized RNA or the input template. T herefore, we f urther
examined whether the purified CSFV NS5B had such a
TNTase activity. With the full-length 3¢ UTR as a template,
TNTase activity was tested i n the presence of four different
radiolabeled ribonucleotide triphosphates to see if there was
any end-labeling activity at the 3¢ terminal of the temperate.
As shown i n Fig. 1D, the radiolabeled products were
detected in all t he reactions containing the NS5B protein
(lanes 1–4) but not in the control experiments containing
fractions isolated from untransformated E. coli lysate (lanes
5–8), i ndicating that the C SFV NS5B protein did have a
TNTase activity.
The 3¢ UTR is believed to be the first entry site for viral
replicases to initiate RNA genome replication. To charac-
terize the binding activity of the CSFV RdRp, estimation of
the binding affinity of t he purified NS5B to the 3¢ UTR was
performed by EM SA. The 603 nucleotide RNA template
consisting of the full-length CSFV 3¢ UTR and the s ubse-

quent coding sequence from the plus-strand genome,
designed as +RNA0 (also see Fig. 4A), was labeled w ith
[
32
P]UTP[aP]. The 701 nucleotide fragment co ntaining t he
full-length minus-strand 3¢ UTR and the subsequent
upstream sequence, designed as –RNA0, was a lso labeled
with [
32
P]UTP[aP]. The purified NS5B proteins at various
concentrations were incubated with a fixed amount of the
radiolabeled +RNA0, and the reaction products were
resolved on a polyacrylamide gel under nondenaturing
conditions. The gel was subjected to autoradiography. As
shown i n F ig. 2A, the amount of RNA–NS5B complex
retarded at the loading wells was in agreement with
increasing amount of purified NS5B protein. At the same
time, the EMSA was carried out under the condition of a
fixed amount of the NS5B and the radiolabeled +RNA0 in
the presence of increasing a mount of competitor, the
unlabeled +RNA0. The EMSA showed that RNA of the
RNA–NS5B complex specifically competed with + RNA0.
These r esults clearly d emonstrate an i nteraction between
CSFV NS5B p rotein and the +RNA0 (Fig. 2B). In t he
same way, the E MSA containing a fixed a mount of the
NS5B and the radiolabeled –RNA0 in the presence of
increasing amounts of c ompetitor, the unlabeled –RNA0
also demonstrated an interaction between CSFV NS5B
protein and the –RNA0 (results not shown).
Mapping of the NS5B-binding site on the CSFV genome

The NS5B protein is the replicase for the C SFV genome.
Therefore, all sites of the full-length CSFV genome could be
bound for the NS5B protein. Some bindings were weaker
and some stronger. Only the stronger bindings might be
efficient to initiate genome replication. To define more
precisely the site of the viral genome essential to efficient
binding of CSFV NS5B protein, a series of c ompetition
experiments were performed with the radiolabeled +RNA0
and various RNA competitors derived from different sites
of the CSFV plus-strand genome. In addition to th e above
+RNA0, these R NA competitors contained plus-strand
5¢ UTR, +RNAr1 (a 375 nucleotide RNA fragment
corresponding to the c oding sequence n ext to 3 ¢ UTR),
and +RNAr2 (an random sequence corresponding to
positions 1245–1700 of the p lus-strand genome). As shown
in Fig. 3A, the complex formed b etween the r adiolabeled
+RNA0 and the NS5B protein was almost completely
abolished by 25-fold molar excess of the unlabeled +RNA0
(lane 5). In contrast, the +5¢ UTR, +RNAr1 and
+RNAr2 had n o effect on the binding (lanes 6 –15). The
fact that the +RNAr1 without 3¢ UTR had no effect on the
complex formed between the radiolabeled +RNA0 con-
taining 3 ¢ UTR and the NS5B p rotein, showed that the s ite
of interaction between them is 3¢ UTR. Moreover, our
results suggest that the NS5B protein bound more 3 ¢ UTR
than other regions of the plus-strand genome.
The Pestivirus genomic replication consists of two
consecutive processes. Replicase first recognizes an d binds
the 3¢ UTR and starts RNA s ynthesis, in which a minus-
RNA is produced with the plus-genomic RNA as a

template. Then, a progeny plus-RNA is produced with the
novel minus-RNA as a template [4]. Therefore, t he specific
cis-element and the structure for direction of a plus-RNA
synthesis might be present in the minus-RNA, as well as i n
the plus-RNA. To investigate the binding activity of the
CSFV NS5B protein on the minus-RNA, the competitive
EMSA was performed with the R NA competitors
1234567
1234567
8
ABNS5B +RNA0
Fig. 2. Formation of the complex between CSFV and +RNA0. (A) The
EMSA was performed with purified NS5B proteins at various con-
centrations (lanes 2–7 for 90, 100, 200, 300, 400, 500 ng, respectively)
incubated with a fi xed amount of the radiolabeled +RNA0. +RNA0
consisted of t he 228 nucleotide full-length viral 3¢ UTR and subse-
quent 375 n ucleotid e co ding re gion. Lane 1 represents f ree + RNA0
probe to which no NS5B protein was added. (B) The EMSA was
carried out with a fixed amount of the NS5B in t he presence of
increasing amount of competitor +RNA0 (lanes 1–8 fo r 0, 0.5, 1 , 5 ,
10, 25, 50, 100 pmol, respectively).
Ó FEBS 2004 Interaction between CSFV NS5B and the genome (Eur. J. Biochem. 271) 3891
derived from the minus-RNA of the g enome. These RNA
competitors were –RNA0, –RNAr1 (a 328 nucleotide
fragment adjacent to the minus-strand 3¢ UTR), –RNAr2
(a random sequence corresponding to positions 2033–2500
of the minus-strand g enome) and minus-strand 5¢ UTR.
Similarly, we found that the –RNA0 competed most
effectively with the labeled RNA for binding of the CSFV
NS5B protein among the competitors, and that –RNAr1,

–RNAr2 and )5¢ UTR had no effect on the binding at all
(results not shown), i ndicating that a n interaction occurred
between the NS5B protein and the native minus-strand
3¢ UTR.
It is known that both the plus-genomic RNA and the
minus-genomic RNA are present in viral host cells when the
replication of positive-se nse RNA virus o ccurs. It is
necessary to compare the binding activities o f the plus-
strand 3¢ UTR–NS5B with that of the minus-strand
3¢ UTR–NS5B. The purified recombinant NS5B was incu-
bated with the radiolabeled –RNA0 in the presence of
unlabeled +RNA0 or unlabeled –RNA0. Interestingly,
RNA0 was stronger than +RNA0 in the interaction with
the N S5B protein (Fig. 3B), i ndicating that NS5B bound
the minus-strand 3¢ UTR was more efficiently than the plus-
strand 3¢ UTR.
Mapping of the specific NS5B-binding sequence
on the 3¢ UTR
From the a bove experimental r esults, it i s d erived that a
specific element recognized by the viral RNA polymerase to
initiate RNA replication might be harbored within the plus-
strand 3¢ UTR a nd the minus-strand 3¢ UTR. To detect
the specific NS5B-binding sequence, a further competition
experiment was conducted. Various RNAs were used as the
competitor, resulting from deletion of the n ative 3 ¢ UTR
with PCR and subsequent in vitro transcription. The plus-
strand genome was first addressed: +RNA1 t o +RNA5
represent, respectively, the RNA templates containing the
3¢ UTR with deletion of ÔCÕ, ÔCCÕ, ÔCCCÕ, ÔCCCGGÕ and the
21 nucleotide fragment at t he 3¢ terminal of the plus-strand

genome (Fig. 4A). The radiolabeled +RNA0 was used as
the substrate for the NS5B protein for t he competition
experiments. Among these RNAs, +RNA1 c ompeted
most efficiently (Fig. 4B, lanes 2–4), followed by +RNA2
and +RNA3 (lanes 5–10). In contrast, +RNA4 and
+RNA5 w ere poor competitors (lanes 11–16). These results
indicated that the CSFV NS5B protein bound the 3¢ UTR
mainly through interaction with the  21 nucleotide bases at
the t erminal. The ÔCCCGGÕ at the 3¢ terminal of the plus-
strand 3¢ UTR was important for binding th e NS5B
protein. Similarly, the binding experiment was p erformed
containing the RNA competitors f rom the mutation of the
minus-strand 3¢ UTR: –RNA1 to –RNA5 represent,
respectively, the RNA templates containing the minus-
strand 3¢ UTR with deletion of ÔCÕ, ÔCATATGÕ, ÔCATA
TGCTÕ, ÔCATATGCTCÕ and the 21 nucleotide fragment
at the 3¢ terminal. We found that –RNA1 was the m ost
efficient competitor, then –RNA4, a nd –RN A5 w as the
poorest among these minus-strand RNA fragments
(Fig. 4 C). Therefore, t he 3¢ ÔCATATGCTCÕ sequence of
3¢ terminal of the minus-strand genome is essential to
binding of the CSFV NS5B protein. The minus-strand
3¢ UTR with deletion of the 21 nucleotide fragment at the
terminal did not interact with the N S5B protein at all.
Characterization of the
cis
-acting sequence at the
plus-strand or minus-strand 3¢ UTR for RNA synthesis
To characterize the cis-acting s equence a t the plus-strand
3¢ UTR for RNA synthesis, RdRp assays containing the

123456
–
+RNA0 +RNAr1 +RNAr2 +5 UTR
'
–
78
12345678
9 1011121314
A
–
–RNA0
–
B
+RNA0
Fig. 3. Mapping of the NS5B-binding site on the CSFV genome.
(A) Competitive EMSA was performed with CSFV NS5B protein and
the
32
P-labeled +RNA0 in absence (lane 2) or the presence (lanes
3–14) of increasing amounts (1, 5, 25 pmol, respectively) of various
competitors (+RNA0, +RNAr1, + RNAr2, and +5¢ UTR, as
indicated). +RNAr1 i s a 375 nucleotide RNA fragment correspond -
ing to the co ding sequence next to 3¢ UTR, and +RNAr2 i s a 456
nucleotide random sequence of coding region . Lane 1 represents free
+RNA0 pro be to which no NS5B protein was added. (B) The results
were obtained from com petitive EMSA with CSFV NS5B protein and
the
32
P-labeled –RNA0 in the presence of unlabe led +RNA0 ( lanes
6–8 for 10, 25, 50 p mol, respectively) or –RNA0 (lanes 3–5 for 10, 25,

50 pmo l, respectively ). –RNA0 consist s of a 373 nucleotide minus-
strand 3¢ UTR and a 328 nucleotide subsequent up stream sequence.
Lanes 1 and 2 represent no N S5B protein and n o unlabeled –RNA0,
respectively.
3892 M. Xiao et al.(Eur. J. Biochem. 271) Ó FEBS 2004
above five mutant RNAs were performed. The CSFV NS5B
protein was incubated with +RNA1, +RNA2, +RNA3,
+RNA4 and +RNA5, respectively, for 2 h in the presence
of radiolabeled CTP. The reaction prod ucts were analyzed
on an agarose g el. As expected, the newly synthesized RNA
from the RNA polymerization containing +RNA1 and
+RNA2 was de tected (Fig. 5A, lanes 1 and 2 ). +RNA3,
+RNA4 and +RNA5 were unable to direct RNA
synthesis (Fig. 5A, lanes 3–5). These results showed that
the three continuous ÔCÕ at the 3 ¢ terminal of the plus-strand
3¢ UTR were essential to the initiation of RNA synthesis.
Similarly, the cis-acting sequence n ecessary for RNA
synthesis on the minus-strand CSFV g enome was examined
as described above. The RNA templates with different
lengths were formed f rom the minus-strand CSFV genome
through PCR and subsequent in vitro transcription. The
RdRp reactions containing these R NA tem plates ( Fig. 4A;
–RNA1, –RNA2, –RNA3, –RNA4 and –RNA5) were,
respectively, performed in t he presence of the CSFV NS5B
protein. It was observed t hat –RNA1 and –RNA2 were
efficient templates a nd yielded a major product (Fig. 5B,
lanes 1 and 2). In contrast, – RNA3, – RNA4 and the RNA
with deletion of the 21 nucleotide fragment at the 3¢ terminal
of the minus-RNA genome did not have any template
activity (F ig. 5A, lanes 3–5), indicat ing that several con-

tinuous nucleotide b ases at the 3¢ ter minal of the minus-
strand CSFV genome were important to initiating synthesis
of the plus-strand RNA.
Comparatively, we found that these results from the
RdRp assays were essentially in agr eement with the data
from the c ompetition EMSA. T aken together, t hese results
suggested that the  21 nucleotide fragment l ocated at the 3¢
end of plus-strand 3¢ UTR might be the first site for the
CSFV NS5B protein to initiate R NA genome replication,
whereas the promoter sequence for NS5B protein to
synthesize the plus-strand RNA was 21 continuous nucleo-
tide bases at the 3 ¢ terminal of the minus-strand C SFV
genome.
Discussion
Using E. coli cells as expression systems, we obtained the
purified full-length CSFV NS5B protein. The NS5B protein
is able to synthesize minus-strand RNA with plus-strand
12345 12345
AB
600
600
Fig. 5. RdRp assays containing various RNA templates with mutant
3¢ UTR. The products from RdRp assays at 37 °C for 2 h containing
the R NA templates with mutant 3¢ UTR w ere loaded onto 1.5%
agarose gel containing 2.2
M
formaldehyde, 40 m
M
MOPS (pH 7.0),
10 m

M
sodium acetate, and 1 m
M
EDTA. ( A) Mutant plus-strand
RNA 3¢ UTR (lanes 1–5; +RNA1 to +RNA5). ( B) RNA templates
with the mutant minus-strand 3¢ UTR (lanes 1–5; –RNA1 to –RNA5).
Numbers to the left refe r to t he position o f RNA co ntainin g 600
nucleotides.
11615141312111023 45 6 789
11615141312111023456789
B
A
CGGCCC +RNA0
–
+RNA1
+RNA2 +RNA3 +RNA4 +RNA5
C
–
–RNA1 –RNA2 –RNA3 –RNA4 –RNA5
CGGCC +RNA1
CGGC +RNA2
CGG +RNA3
C +RNA4
+RNA5
ACCTCGTATAC –RNA0
ACCTCGTATA –RNA1
ACCTC –RNA2
ACC –RNA3
AC –RNA4
–RNA5

Fig. 4. Map ping of t he specific NS5B-binding s e quenc e on t he plus-
strand 3¢ UTR or minus-strand 3¢ UTR. (A) Various RNA t emplates
containing the wild-t ype an d the m utant plus- strand 3 ¢ UTR or minus-
strand 3¢ UTR. )RNA1 to )R NA5 represe nt, respect ively, the RNA
templates containing the plus-strand 3¢ UTR with deletion of ÔCÕ, ÔCCÕ,
ÔCCCÕ, ÔCCCGGÕ and t he 21 nucleotide fragment at the 3¢ terminal.
–RNA1 to –RNA5 represent, respectively, the RNA templates con-
taining the m inus-strand 3¢ UTRwithdeletionofÔCÕ, ÔATATGÕ,
ÔATATGCTÕ, ÔATATGCTCÕ and the 21 nucleotide fragment at the 3 ¢
terminal. (B) The c o mpetitive EMSA was preformed with CS FV
NS5B protein and the
32
P-labeled +RNA0 in the absence (lane 1) or
presence (lanes 2–16) of increasing amount (10, 25, 50 pmol) of various
competitors (+ R NA1 to + RNA5). (C) The competitive EMSA w as
preformed with CSFV NS5B protein and the
32
P-labeled -RNA0 in
absence (lane 1) or the presence (lanes 2–16) of increasing amount (10,
25, 50 pmol, respectively) of various competitors (– RNA1 to –RNA5,
as indicated).
Ó FEBS 2004 Interaction between CSFV NS5B and the genome (Eur. J. Biochem. 271) 3893
native CSFV 3¢ UTR a s a template and to synthesize plus-
strand RNA with minus-strand CSFV native 3¢ UTR as a
template. Under RdRp assay conditions, the protein
operates highly processively on these RNA templates up
to several hundred nucleotides long. Therefore, the CSFV
NS5B protein from prokaryotic cell expression systems is
demonstrated to have RdRp activity, as reported previously
[7]. This enzyme does not require an exogenous primer, and

can recognize t he specific sequence for initiation of RNA
synthesis. In addition to the RdRp activity, we showed that
the CSFV NS5B protein possessed the TNTase activity. A
TNTase protein does not proceed in elongating the primer
after a ddition of the first residue, and is able to catalyze the
addition of a single nucleotide residue to the 3¢ terminal of
an RNA template, which was observed in our preparation
of purified CSFV NS5B p rotein. In some p revious reports,
HCV NS5B has been shown to possess the T NTase activity
in addition to the RdRp activity [14,15,31], whilst in others
it has not [17]. It has been observed that the TNTase activity
is associated with BVDV NS5B protein [31]. Although
CSFV NS5B proteins have been demonstrated to have the
RdRp activity, little is known a bout whether t hese enzymes
possess TNTase activity before the current rep ort. Th e fact
that the CSFV NS5B protein was shown to have the
TNTase activity in addition to an RdRp activity in this
research supports the proposal that a copy-back mechanism
is associated with initiation of RNA synthesis. It is assumed
that the synthesis of a template-independent 3¢ extension is
first performed by the TNTase activity, f ollowed b y the
looping back and priming. A copy-back mechanism has
been reported to be associated with initiation of RNA
synthesis in HCV and BVDV [12,14,15], however, it has
recently been found that the mechanism of de novo initiation
is preferred f or the HCV NS5B pr oteins [17,19–21]. This
mechanism has been observed in BVDV NS5B for initiation
of RNA synthesis [21]. It was also found that both the copy-
back mechanism and the de novo mechanism for initiation
of RNA synthesis m ight be p resent in the NS5B protein of

CSFV and BVDV [7,13]. The fact that the CSFV NS5B has
a T NTase a ctivity, toget her with other data i n t his r esearch
and previous reports, increases this possibility that two
mechanisms, copy-back a nd de novo, are compatible with
each other in the CSFV viral genomic replication.
To characterize the i nteraction between the CSFV NS5B
protein and different sites of the genome, competitive
electrophoretic mobility shift assays were performed.
Firstly, the plus-strand genome was addressed. The NS5B
protein was incubated in the presence of the r adiolabeled
3¢ UTR and different site sequences as competitor. It was
observed that a specific complex was formed between CSFV
NS5B protein and its full-length 3¢ UTR. The 5¢ UTR
formed the complex poorly as the 3¢ UTR was a superior
competitor relative to the 5¢ UTR. The rando m sequence of
coding region had no effect o n the formation of the
complex. Our conclusion is consistent with that drawn from
a similar study by using EMSA, in which specific interaction
between the H CV NS5B protein and the noncoding region
oftheviralgenomewasalsoaddressed[32].Inour
competitive E MSA towards the minus-strand genome, the
minus-strand 3¢ UTR was the strongest c ompetitor, fol-
lowed b y t he random sequence of c oding region and t he
minus-strand 5¢ UTR, similar to the competitive experiment
towards the plus-genome. Interestingly, when the plus-
strand 3¢ UTR and the minus-strand 3¢ UTR were used at
the s ame t ime, the NS5B p roteins bound to the minus-
strand 3¢ UTR more strongly than to the p lus-strand
3¢ UTR. Although we have not yet found sufficient
evidence, it is proposed that th e activity of the minus-strand

3¢ UTR is stronger than the plus-strand 3¢ UTR in
initiation of RNA synthesis. Indeed, it i s observed that a
greater amount of plus-strand RNA than minus-strand
RNA is d etected when the BVDV was replicating in its
native cells [4]. This obse rvation was reported in the ce lls
harboring the HCV RNA replicon, in which the plus-strand
RNA was more abundant than the minus-strand RNA [33].
These results agree with the fact that the HCV RNA-
dependent RNA polymerase replicates in vitro the 3¢
terminal region of the minus-strand viral RNA more
efficiently than the 3¢ terminal region of the plus-RNA
[26]. Further, we found that the mutated plus-strand
3¢ UTR with deletion of 5 or 21 nucleotide bases at the 3 ¢
terminal lost binding activity, and that the m utated minus-
strand 3¢ UTR with deletion of a 9 or 21 nucleotide sequence
at the 3¢ terminal also did not have the b inding activity,
indicating that the CSFV NS5B p rotein binds the 3¢ UTR
mainly through interaction with the s everal nucleotide bases
at the t erminal of the 3¢ UTR. Therefore, the 3¢ UTR,
specifically the 3 ¢ terminal region, is very important fo r
binding of the CSFV NS5B protein, whereas the sequences
of the c oding region have no effect on the binding at all,
irrespective of w hether it is plus-strand genome or not; t his
is not in agreement with t he previous observation from
HCV in which the partial NS5B-coding sequence and
subsequent partial 3¢ UTR are the necessary sites for
binding o f NS5B protein [25]. But, our data is consistent
with Cui and his colleagues’ reports that the 3 ¢ end o f
encephalomyocarditis virus is involved in the binding of
RdRp [34,35]. In addition to NS5B protein, other viral

proteins are a ble to b ind the 3¢ UTR, e.g. the H C V NS3
protein can bind the 3¢ UTR in vitro and displayed a
protease and a helicase activity [36]. Some cellular proteins
arealsoabletobind3¢ UTR, such as the heterogeneous
nuclear ribonucleoprotein C [37] and r ibosomal proteins
[38].
To analyze the function of the NS5B-binding sequence i n
the initiation o f RNA synthesis, we performed the RdRp
assays, i n which the mutated 3¢ UTRs with deletions of
different l engths at the 3¢ terminal of the C SFV p lus-strand
genome were addressed. The plus-strand 3¢ UTR with
deletion of 3, 5 or 21 nucleotide bases at the 3¢ terminal was
observed to lose the activit y for i nitiation o f minus-RNA
synthesis. When the mutated minu s-strand 3¢ UTRs with
deletions of different lengths at the 3¢ terminal were used in
the RdRp assays, similar results were observed, i.e. the
minus-strand 3¢ UTR with 8, 9 or 21 nucleotide bases
deleted from its 3 ¢ terminal had no template a ctivity.
Together with the above binding data, it is s hown that the
sequence approximately b etween position 1 and 2 1 at the
CSFV genome, irrespective of whether it is plus-strand
genome or not, might be the cis-acting signals for i nitiation
of the C SFV R NA synthesis, which is compatible w ith ou r
earlier conclusion that the sequence of the 3¢ terminal of t he
3¢ UTR is essential to initiation of RNA synthesis [7,9]. In
this study, we noted that the template activity o f t he RNAs
3894 M. Xiao et al.(Eur. J. Biochem. 271) Ó FEBS 2004
was well in agreement with their binding activity, which is
consistent with the earlier reports that the t emplate activity
of RNAs for Qb replicase i s correlated with their binding

affinity [39]. A similar conclusion has also been drawn from
the HCV [25]. Our results differ f rom a nother study in
which t he template activity of RNAs for the HCV N S5B i s
inversely correlated with the binding activity of these RNAs
to the N S5B [15]. This discrepancy may be partially due to
the f act t hat homopolymeric RNAs were used as templates
in that study and we used heteropolymeric RNAs as
templates. Previous studies have demonstrated specific
sequences and s tructures at the 3¢ terminal of plus-strand
RNA v iruses involving the binding of RNA polymerase and
initiation of replication, e.g., a 3¢ terminal 98 nucleotide
X-region of HCV [25,32], the secondary structure and the
single-strand region a t the 3¢ end of BVDV 3¢ UTR [3], a
nonbase-paired 3¢ ÔACCÕ sequence o f a tRNA-like s tructure
of turnip yellow mosaic v irus [40], a psudoknot structure of
polioviruses [41], and a stem-loop structure of turnip crinkle
virus [ 42]. Our experiments show ed that the 3¢ ÔCCCGGÕ
sequence at the plus-strand 3¢ UTR and the 3¢ ÔCATA
TGCTCÕ fragment at the minus-strand 3¢ UTR is essential
to the CSFV R NA synthesis. The 3¢ UTR with the above
deletion has no template activity, and also no b inding
activity to NS5B. The predicted secondary structure of these
mutant 3¢UTRs is not more stable than that of their wild-
type equivalent [7]. The f act that an internal single-strand
region is necessary for binding of HCV NS5B protein and
initiating RNA s ynthesis fuels the possibility that internal
initiation rather than initiation from the very 3¢ terminal is
associated with HCV [25], in contrast to our conclusion that
the mechanism of initiation from the very 3 ¢ termina l of
3¢ UTR might be one of peculiarities of CSFV replication

because the sequence of the 3¢ terminal of 3¢ UTR is
necessary fo r binding of NS5B protein and initiation of
RNA synthesis. Our previous report shows that the CSFV
RdRp originating from expression in prokaryotic cells has
high template specificity. Replacement and deletion in the
middle part of the viral 3¢ UTR has little influence on
initiation of RNA synth esis. The 3¢ terminal of the 3 ¢ UTR
is much more important to initiation of RNA synthesis than
its m iddle p art [7]. The importance of the 3¢ terminal of the
3¢ UTR is reinforced in this report.
Acknowledgements
This work was s upported by National Natural S cience Foundation of
China (30170214) and Science and Technology Foundation of
Shanghai Higher Education.
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