De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase Guang-Hui Yi, Chu-Yu Zhang, Sheng Cao, Hai-Xiang Wu and Yi Wang Institute of Virology, Col
Trang 1De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase
Guang-Hui Yi, Chu-Yu Zhang, Sheng Cao, Hai-Xiang Wu and Yi Wang
Institute of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
Classical swine fever virus nonstructural protein 5B (NS5B)
encodes an RNA-dependent RNA polymerase, a key
enzyme of the viral replication complex To better
under-stand the initiation of viral RNA synthesis and to establish
an in vitro replication system, a recombinant NS5B protein,
lacking the C-terminal 24-amino acid hydrophobic domain,
was expressed in Escherichia coli The truncated fusion
protein (NS5BD24) was purified on a Ni-chelating HisTrap
affinity column and demonstrated to initiate either
plus-or minus-strand viral RNA synthesis de novo in a
primer-independent manner but not by terminal nucleotidyle
transferase activity De novo RNA synthesis represented the
preferred mechanism for initiation of classical swine fever
virus RNA synthesis by RNA-dependent RNA polymerase
in vitro Both Mg2+and Mn2+supported de novo initiation, however, RNA synthesis was more efficient in the presence
of Mn2+than in the presence of Mg2+ De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mMGTP, and a 3¢-terminal cytidylate on the viral RNA template was preferred for de novo initiation Furthermore, the purified protein was also shown, by North-Western blot analysis, to specifically interact withthe 3¢-end of bothplus-and minus-strbothplus-and viral RNA templates
Keywords: classical swine fever virus; RNA-dependent RNA polymerase; nonstructural protein 5B; de novo RNA synthesis; RNA-binding activity
Classical swine fever virus (CSFV) is a small enveloped
positive-strand RNA virus classified in the genus of
Pestivirus, which also comprises bovine viral diarrhea virus
(BVDV) and border disease virus (BDV) Together with the
genera Flavivirus and Hepacivirus, they form the family
Flaviviridae[1,2] The genomic RNA, 12.3 kb in length,
contains a single long ORF encoding a polyprotein of
3898 amino acids which is flanked by 5¢- and 3¢-UTRs [3]
The 5¢-UTR contains an internal ribosomal entry site
(IRES) for cap-independent translation of the viral
poly-protein [4,5], whereas the 3¢-UTR may contain replication
signals involved in minus-strand RNA synthesis, as in
BVDV [6] The polyprotein is processed, co- and
post-translationally, into 12 polypeptides by viral and cellular
proteases The order of polypeptides is NH2-Npro-C-Erns
-E1-E2-p7-NS2-NS3-NS4A-NS4B-NSA-NS5B-COOH [7]
Npro, a nonstructural autoprotease, can release itself from
its precursor, but is not necessary for viral replication in cell
culture [8] Nuclecapsid protein C, and glycoproteins Erns,
E1 and E2, represent four structural proteins, and form the
capsid and envelope of the virion, respectively The others
are nonstructural proteins (NS) [3] Most of the NS are speculated to be components in the viral replication cycle Among them, NS3 is a multifunctional enzyme and responsible for functions associated withthe replication and biotype of cytopathogenicity in cell culture [9] The last viral protein (NS5B), at the C terminus of the polyprotein, is
a key component responsible for the replication of viral RNA genome It also contains motifs shared by RNA-dependent RNA polymerases (RdRps), suchas the Gly– Asp–Asp (GDD) motif, which is highly conserved among RdRps [10] and has been demonstrated to possess RdRp activity in insect cells [11,12] and porcine kidney cells (PK-15) [13] It is believed that certain enzymatic functions of NS3 and NS5B may play an important role during the replication of viral RNA
The replication of the CSFV genome is generally thought
to be similar to other positive-strand RNA viruses: synthesis
of complementary minus-strand RNA withthe plus-strand genomic RNA as template, and subsequent synthesis of the progeny RNA withthe minus-strand RNA as template Thus, the 3¢-end of bothplus- and minus-strand RNAs may contain the cis-acting elements, suchas promoter or enhancer, involved in the initiation of viral RNA synthesis
by RdRp Although several infectious cDNA clones of CSFV have been developed [14–18], facilitating the research
of cytopathogenicity, replication and function of viral proteins by reverse genetic approachin the cell culture system [8,9,19], another method to study CSFV replication would be to work towards identification of the cis-acting elements at the 3¢-end of bothplus- and minus-strand RNAs and possibly the viral or cellular proteins that interact with
it to form a replication complex for initiating viral RNA synthesis in vitro At present, the molecular mechanism of
Correspondence to C.-Y Zhang, Institute of Virology, College of Life
Sciences, Wuhan University, Wuhan 430072, China.
Fax: + 86 27 87883833, Tel.: + 86 27 87682833,
E-mail: avlab@whu.edu.cn
Abbreviations: ALP, alkaline phosphatase; BVDV, bovine viral
diarrhea virus; CSFV, classical swine fever virus; DIG, digoxin;
RdRp, RNA-dependent RNA polymerase; IRES, internal
ribosomal entry site; NS5B, nonstructural protein 5B; TNTase,
terminal nucleotidyle transferase.
(Received 23 September 2003, accepted 24 October 2003)
Trang 2initiating CSFV RNA synthesis is not well understood.
Previous reports have shown that the recombinant CSFV
NS5B (expressed in insect cells) catalyzed RNA synthesis
was strictly primer-dependent and that intramolecular
priming copy-back synthesis represented the preferred
mechanism for initiation of RNA synthesis [11] However,
the activity of the cellular terminal nucleotidyle transferase
(TNTase) was also demonstrated to be present in the
cytoplasmic extracts of insect cells [20,21] The cellular
TNTase could add extra nucleotides to the 3¢-terminus of
the RNA template and might serve as primer for
template-primed copy-back synthesis Moreover, evidence was
obtained that the TNTase activity associated with the
hepatitis C virus NS5B might be the result of a
contamin-ating cellular protein present in minute amounts in the
enzyme preparation [21]
To better understand the initiation of CSFV RNA
replication, we expressed and purified a recombinant
NS5BD24 fusion protein from Escherichia coli BL21
(DE3) The fusion protein was demonstrated to have the
ability to initiate de novo either plus- or minus-strand viral
RNA synthesis in a primer-independent manner and to
specifically interact withviral RNA templates This in vitro
RdRp assay will be useful for using to study the sequences
and proteins required for the initiation of CSFV RNA
synthesis
Materials and methods
Plasmid constructs
The plasmid pGEM5b, containing full-length CSFV
(Shi-men strain) NS5B, was constructed as described previously
[13] The NS5B fragment lacking the C terminal 24 amino
acids (NS5BD24) was PCR amplified withthe following
primer pair – NS5BFor and NS5BRev – from pGEM5b
(Table 1) A polyhistidine tag (GSHHHHHH) was
intro-duced at the C terminus to facilitate purification of the
NS5BD24 protein After purification, the PCR products
were digested with NcoI and BglII and then inserted into
the NcoI/BamHI sites of vector pET-28a (Novagen) The
resulting expression vector, pET-NS5BD24, which was
driven by the T7 RNA polymerase promoter, was
trans-formed into E coli DH5a Site-directed mutagenesis of
GDD to GAA, containing the double substitution of both
Asp448 and Asp449 to alanine, was carried out by
overlapping PCR The N terminal 1370 bp fragment was
amplified by NS5BFor and NS5Bm2, and the C terminal
780 bp fragment was amplified by NS5Bm1 and NS5BRev
Then, the two fragments were purified and combined to
generate the mutant NS5BD24GAA using the outer primers
NS5BFor and NS5BRev After modification with NcoI
and BglII, the mutant fragment was subcloned into vector
pET-28a Transformants were analyzed by restriction
enzyme mapping and confirmed by the dideoxy sequencing
method
Expression and purification of recombinant CSFV
NS5BD24
E coliBL21 (DE3), transformed witheither pET-NS5BD24
or pET-NS5BD24GAA, was grown in Luria–Bertani (LB)
medium, at 37C, to an attenuance (D) at 600 nm of 0.6– 0.8 Then, the temperature was lowed to 18C, and protein expression was induced for 20 hby the addition of 0.4 mM
isopropyl thio-b-D-galactoside (IPTG) The cell pellet obtained from 500 mL of culture was resuspended in
30 mL of binding buffer containing 20 mMsodium phos-phate, pH 8.0, 500 mM NaCl, 20 mM imidazole, 10 mM
b-mercaptoethanol, 20% glycerol, 1% Triton-X-100, 1 mM
phenylmethanesulfonyl fluoride, and 10 lgÆmL)1lysozyme Then 20 lL of DNase I (Takara) was added to the suspension for 30 min at room temperature The lysates were sonicated on ice to reduce viscosity, and any insoluble materials were removed by centrifugation at 13 000 g for
15 min The clear supernatant was applied to a 1-mL Ni-chelating HisTrap affinity column (Amersham) pre-equilibrated withthe binding buffer containing 50 mM
imidazole The bound protein was then eluted stepwise withelution buffer (20 mM sodium phosphate, pH 8.0,
500 mM NaCl, 5 mM b-mercaptoethanol, 20% glycerol, 0.2% Triton-X-100) containing a gradient imidazole con-centration from 150 to 450 mM Th e fractions were monitored by SDS/PAGE and staining withCoomassie Brilliant Blue R250 Then, the His-tagged protein peaks were collected and dialyzed against buffer (20 mMTris/HCl
pH 8.0, 500 mM NaCl and 20% glycerol), followed by storage at)40 C in small aliquots The concentration of purified protein was determined by the Bradford method using BSA as a standard
SDS/PAGE and Western blot Protein fractions from the HisTrap affinity column were separated by 12% SDS/PAGE and electrotransferred to a nitrocellulose membrane The membrane was blocked with
Table 1 Sequence of the primers used in this study The T7 polymerase promoter sequence is shown in italics The mutated nucleotides are shown in bold and the additional polyhistidine amino acid sequences are underlined.
Primers Sequence (5¢ )3¢)
GCTGCCATTGTACCTGTCTGCCCCTT
5¢-UTRfor GTATACGAGGTTAGTTCATTC 5¢-UTRfor1 CTATACGAGGTTAGTTCATTC 5¢-UTRfor2 TTATACGAGGTTAGTTCATTC 5¢-UTRfor3 ATATACGAGGTTAGTTCATTC 5¢-UTRrev TAATACGACTCACTATAGGGTGCCATGAA
CAG 5¢-UTRrev2 GTGCCATGAACAGCAGAGATTTTTATAC
3¢-UTRfor TAATACGACTCACTATAGCGCGGGTAAC 3¢-UTRfor2 GCGCGGGTAACCCGGGATCTGAA
3¢-UTRrev1 CGGCCGTTAGGAAATTACCTTAGTC 3¢-UTRrev2 TGGCCGTTAGGAAATTACCTTAGTC 3¢-UTRrev3 AGGCCGTTAGGAAATTACCTTAGTC
Trang 33% BSA in NaCl/Pi and treated withrabbit anti-swine
serum infected with CSFV Alkaline phosphatase
(ALP)-conjugated goat anti-(rabbit IgG) was used as the secondary
antibody After washing three times with NaCl/Pi
contain-ing 0.1% Tween-20, membrane-bound antibodies were
detected withNitro Blue tetrazolium/5-bromo-4-ch
loro-indol-2-yl phosphate
Preparation of RNA templates and RNA labeling
RNA templates were prepared by in vitro transcription The
plasmid T6-1, containing the full-length CSFV 3¢-UTR,
and the plasmid pGEM61s-2, containing the full-length
5¢-UTR, were constructed as previously reported [18] The
DNA fragment of th e 3¢-end of the plus-strand RNA
(+)3¢-UTR was amplified using primers 3¢-UTRfor and
3¢-UTRrev from T6-1; and the 3¢-end of the minus-strand
RNA (–)IRES, which is complementary to the CSFV
5¢-UTR, was amplified from pGEM61s-2 using the primers
5¢-UTRfor and 5-¢UTRrev To generate (+)3¢-UTR
mutants withsubstitution of the 3¢ terminus cytidylate with
G, A and T, PCR was performed withsense primer
3¢-UTRfor and antisense primers 3¢-UTRrev1, 3¢-UTRrev2
or 3¢-UTRrev3, respectively, using T6-1 as template For
(–)IRES mutants, PCR was perfomed using the sense
primers 5¢-UTRfor1, 5¢-UTRfor2 or 5¢-UTRfor3 and
anti-sense primer 5¢-UTRrev from pGEM61s-2 All the PCR
amplifications were performed using pfu DNA polymerase
(MBI), and the DNA fragments were recovered using a
DNA purification kit The T7 RNA polymerase promoter
sequence was introduced into the primers 3¢-UTRfor and
5¢-UTRrev to initiate RNA synthesis In vitro transcription
was carried out withT7 RNA polymerase, according to the
manufacturer’s instructions (Promega) After a 2
hincuba-tion at 37C, the DNA templates were digested twice with
RNase-free DNase I The RNA transcripts were extracted
with acid phenol/chloroform (1 : 1, v/v), followed by
precipitation withtwo volumes of ethanol and 0.4Msodium
acetate The precipitated RNA was dissolved in diethyl
pyrocarbonate-treated water and the RNA concentration
determined by measuring the absorbance (A) at 260 nm
Occasionally, RNA transcripts were further purified by 6%
PAGE containing 7Murea The gel fragment containing the
RNA was excised and incubated overnight in 10 mMTris/
HCl, pH 7.5, 25 mMNaCl, 1 mMEDTA After elimination
of polyacrylamide, the RNA was precipitated by ethanol
The 3¢-hydroxyl group of RNA transcripts was blocked by
sodium periodate, as described previously [22] Ten
micro-grams of RNA transcript was dissolved in 100 lL of 50 mM
sodium acetate After the addition of 25 lL sodium
perio-date (100 mM), the mixture was incubated for 1 h at room
temperature, then phenol/chloroform extracted and ethanol
precipitated Residual sodium periodate was removed by
several washes using 70% ethanol
For preparation of digoxin (DIG)-labeled RNA probes
or templates, the DNA fragments of (+)3¢-UTR and
(–)IRES were used as templates for in vitro transcription
RNA labeling was performed according to the instruction
manual supplied with the DIG RNA labeling kit of Roche
Molecule Biochemicals The mixtures were incubated for
2 hat 37C, and DNA templates were removed by
digestion withRNase-free DNase I
In vitro RdRp The in vitro RdRp standard assay was performed in a total volume of 50 lL containing 20 mM Tris/HCl, pH 8.0,
5 mM MgCl2, 5 mM MnCl2, 2 mM dithiothreitol, 50 mM
NaCl, 0.25 mMof eachNTP, 0.3 lg of RNA template and
0.1 lg of purified protein The reaction mixtures were incubated at 25C for 2 hand stopped by the addition of
20 mMEDTA The RNA products were extracted with acid phenol/chloroform (1 : 1, v/v) followed by ethanol precipi-tation Then, the precipitates were dissolved with either
20 lL of diethyl pyrocarbonate-treated water or denaturing buffer (see below)
Northern blot analysis The precipitated RdRp products were dissolved in a denaturing buffer containing 95% formamide, 10 mM
EDTA, 20 mM Tris/HCl, pH 8.0, at 100C for 5 min, and then separated by PAGE (8% gel containing 7Murea)
in 1· Tris/borate/EDTA (TBE) buffer After electrophor-esis, the gels were transferred to a positively charged nylon membrane (Hybond) and electroblotted for 4 hat 4C The membrane was dried for 2 h at 80C and exposed to ultraviolet irradiation (254 nm) for 2 min at 0.12 JÆsq cm)1 Hybridization was performed overnight, at 68C, in 10 mL
of a solution containing 50% formamide, 2% blocking reagent (Roche), 5· NaCl/Cit and 20 lgÆmL)1 yeast tRNA, together with the appropriate DIG-labeled RNA transcripts The excess probes were eliminated gradually by washing the membrane from low stringency (2· NaCl/Cit, 0.2% SDS) to high stringency (0.1· NaCl/Cit, 0.1% SDS)
at 68C Then, the bound RNA was treated with ALP-conjugated anti-DIG Ig (1 : 5000) in dilution buffer (1· blocking reagent in 0.1M maleic acid buffer, pH 7.0) for 30 min The reaction complexes were visualized using Nitro Blue tetrazolium/5-bromo-4-chloroindol-2-yl phos-phate, according to the manufacturers of the DIG RNA detection kit (Roche)
RT–PCR and real-time quantitative RT–PCR The strand-specific oligodeoxynucleotide primers, 5¢-UTR-rev2 and 3¢-UTRfor2, complementary to the synthesized plus- and minus-strand RNAs, were used for RT-catalyzed cDNA synthesis, followed by PCR amplification of cDNAs Six microlitres of dissolved RNA was subjected to RT with M-MLV according to the manufacturer’s instructions (Promega) PCR was performed with5¢-UTRfor and 5¢-UTRrev2 primers for detection of synthesized plus-strand RNA For detection of synthesized minus-plus-strand RNA, 3¢-UTRfor2 and 3¢-UTRrev primers were used The synthesized RNA was real-time quantified using the TaqMan assay, according to McGoldrick et al [23] and Cheng et al [24], withsome modifications A set of 5¢-UTR-specific primers, P1 (forward) and P2 (reverse), were used to amplify a 222-bp fragment of the 5¢-UTR (nucleotides 95–316) The fluorogenic probe was designed with the following sequence: 5¢-TACAGGACAGTCGTCAGTAG TTCGACGTGA-3¢ RNA quantification was performed as follows: the precipitated RNA was reverse transcribed to cDNA using P as a primer, then 2 lL of cDNA was used
Trang 4as the template for amplification in a 20-lL volume
containing 15 pmol of eachprimer, 4.5 mMMgCl2, 1 unit
of Taq polymerase (MBI) and 3 pmol of TaqMan probe
(Alpha) The PCR mixtures were placed in a thermocycler
(Corbett) and subjected to 45 cycles of the following
reaction parameters: denaturation at 95C for 30 s,
annealing at 60C for 30 s and extension at 72 C for 45 s
RNA-binding activity by North-Western blot
The protein samples were resolved by SDS/PAGE (10%
gel) and transferred to nitrocellulose membrane
Mem-brane-bound proteins were renatured in buffer A (10 mM
Tris/HCL, 50 mMNaCl, 5% glycerol, 5 mMMgCl2, 0.1%
Triton-X-100, pH 7.8) containing 5 mgÆmL)1 BSA and
1 mM dithiothreitol, at room temperature for 4 h After
washing with buffer A, membranes were transferred to
RNA binding buffer (10 mMTris/HCl, 100 mMNaCl, 5%
glycerol, 5 mMMgCl2, 0.2 mMdithiothreitol, 0.1%
Triton-X-100, 20 lgÆmL)1tRNA, 0.5 mgÆmL)1BSA, pH 7.8) One
microlitre of DIG-labeled RNA was added and the
membranes were incubated for 2 hwithgentle shaking
Membranes were washed twice with RNA binding buffer,
without dithiothreitol and tRNA, and then the bound RNA
was treated withALP-conjugated anti-DIG Ig (1 : 5000)
RNA–protein complexes were visualized using Nitro Blue
tetrazolium/5-bromo-4-chloroindol-2-yl phosphate
Results and discussion
Expression and purification of bacterial recombinant
NS5BD24 and its mutant, NS5BD24GAA
To isolate the function of CSFV NS5B from other viral
and cellular proteins and to establishan in vitro replication
system for studying the initiation of viral RNA replica-tion, NS5B protein was expressed in an E coli system Earlier attempts to express and purify the full-length NS5B had been hampered as a result of the poor solubility Sequence analysis and analysis of the hydro-pathy profile of CSFV NS5B revealed that there was a conserved hydrophobic domain at the C terminus of different CSFV strains (Fig 1A) Prompted by reports that removal of the C terminal hydrophobic domain of the RdRps of hepatitis C virus [25–27], hepatitis G virus [28], and BVDV [29] could significantly improve the solubility of the protein expressed in E coli, th e C terminal 24 amino acids of NS5B were deleted and NS5BD24 was inserted into the pET-28a vector To facilitate protein expression and purification, additional Met–Glu residues were introduced at the N terminus for initiating translation, and a polyhistidine epitope tag (GSHHHHHH) was introduced to the C terminal of NS5BD24 for affinity purification The fusion protein, of
75 kDa, was obtained withan imidazole elution gradi-ent of 150 to 250 mM(Fig 1B) The protein was identified
as the recombinant NS5B by Western blot analysis using CSFV-infected pig serum as primary antibody (Fig 1C)
To maximize the amount of soluble protein, expression was performed at a low temperature (18C) and the cell pellet was resuspended in a nonionic detergent (1% Triton-X-100) in combination witha highconcentration
of salt (500 mM) and glycerol (20%) We succeeded in recovering 2 mg of soluble protein from 1 L of E coli culture The other mutant protein, NS5BD24GAA, was expressed and purified in parallel to the NS5BD24 protein The sufficient amounts of soluble protein thus obtained provided the basis for further studying the characteriza-tion of the enzyme and for the development of an in vitro replication system
Fig 1 Expression and purification of classical
swine fever virus (CSFV) NS5BD24 and
NS5BD24GAA fusion proteins from
Escheri-chia coli (A) Hydropathy profile (Kyte and
Doolittle) of CSFV NS5B NS5B contains a
highly hydrophobic region at the C terminus.
(B) Proteins were expressed and purified as
described in the Materials and methods.
Fractions of sample eluted from the HisTrap
affinity column by a concentration gradient of
imidazole were separated by SDS/PAGE
(12% gel) and stained withCoomassie
Brilli-ant Blue Lane M, molecular mass markers;
lanes 1–4, eluted with150 m M imidazole
buf-fer; lanes 5–8, eluted with250 m M imidazole
buffer; lanes 9–11, eluted with350 m M
imi-dazole buffer (C) Western blot analysis of the
purified protein Lane 1, NS5BD24 protein;
lane 2, pET-28a vector as a negative control;
lane 3, NS5BD24GAA protein.
Trang 5NS5BD24 fusion protein possessed RdRp activity
To determine whether the truncated NS5B (NS5BD24)
protein could direct a viral-specific sequence for initiation of
RNA synthesis in vitro, th e 3¢-end of plus-strand (+)3¢-UTR
and minus-strand (–)IRES RNA transcripts were used as
templates because they were believed to contain cis-acting
promoters for initiating viral RNA synthesis As T7 RNA
polymerase was able to elongate self-complementary RNA
templates by template-directed RNA synthesis during
in vitro transcription [30], both(+)3¢-UTR and (–)IRES
RNA transcripts were purified before being used as
templates RNA templates were loaded onto the same gel
as the marker and were visualized by silver staining The
RNA products synthesized by CSFV NS5BD24 were
separated by denaturing PAGE (8% gel) and detected
using a Northern blot assay As shown in Fig 2 (lane 3), the
purified NS5BD24 was able to synthesize either the
plus-strand or minus-plus-strand RNA products from the respective
templates The predominant RNA products migrated
similarly to the respective RNA templates (373 nucleotides
for synthesized plus-strand RNA and 228 nucleotides for
synthesized minus-strand RNA) When (–)IRES was used
as a template, a very small amount of high molecular weight
RNA products was also observed However, no RNA
products were obtained in the absence of purified protein or
RNA templates, indicating that purified NS5BD24 was not
contaminated withT7 RNA polymerase or RNA/DNA
that could serve as a template Furthermore, mutation of
the conserved motif, GDD, to GAA almost abolished RNA
synthesis (Fig 2, lane 4), similar to the reports for hepatitis
C virus RdRp [31,32] These results showed that purified
NS5BD24 fusion protein, lacking the C terminal 24 amino
acids, possessed RdRp activity in vitro and that the C
terminal hydrophobic domain was not necessary for RdRp activity
Evidence ofde novo RNA synthesis with either
plus-or minus-strand viral RNA as template For the positive strand RNA viruses, the mechanisms of initiating viral RNA synthesis are rather different Poliovi-rus has been shown to use a uridylylated protein as primer (protein-primed) to initiate RNA synthesis [33] Phage Qb initiated de novo RNA synthesis [34], whereas rabbit hemorrhagic disease virus (RHDV) initiated RNA synthesis
by using a template-primed copy-back mechanism [35] Besides copy-back synthesis, Dengue virus RdRp was also demonstrated to be capable of de novo initiation of RNA synthesis [36] Previous work has shown that crude extracts
of recombinant CSFV NS5B expressed in insect cells could produce two RNA products using D-RNA (an mRNA of the liver-specific transcription factor DCoH) as template One product, which was identical in size to the input RNA template, might have resulted from a TNTase activity, while the other was determined to be a double-stranded hairpin dimer RNA synthesized by a copy-back mechanism [11] However, our work found that the predominant RNA products were template-sized, and thus the question was whether the template-sized RNA products were generated through de novo RNA synthesis As the template-sized RNA could be detected by complementary probes, and RNA polymerization required all four ribonucleotides as substrates (data not shown), it seemed that the template-sized RNA products probably represented RNA synthes-ized by a de novo initiation mechanism but should not result from a terminal transferase activity If RNA products were synthesized by TNTase activity, it would have the same polarity as the input RNA template [37] To provide further evidence of de novo RNA synthesis, the 3¢-hydroxyl group
of RNA templates were blocked by treatment withsodium periodate The migration patterns of RNA products are shown in Fig 3A, indicating that the template-sized RNA products were truly synthesized by de novo initiation, but not by the 3¢-end elongation copy-back synthesis It should
be pointed out that a very small amount of high molecular weight RNA products were still observed with 3¢-blocked (–)IRES as template, possibly because the polymerase used the nascent RNA as template for additional rounds of RNA synthesis Furthermore, we performed RT using strand-specific oligodeoxynucleotides as a primer that could anneal only to the 3¢-terminus of either synthesized plus- or minus-strand RNA products, followed by PCR amplification The amplified fragments were analyzed by agarose gel electro-phoresis and stained by ethidium bromide As shown in Fig 3B, the expected sizes of DNA fragments (373 nucleo-tides for new synthesized plus-strand and 228 nucleonucleo-tides for the minus-strand) were observed, verifying that tem-plate-sized RNA products were initiated de novo from the 3¢-terminus of the template but not by premature termin-ation or internal inititermin-ation, as suggested in reports for tomato bushy stunt virus, cucumber necrosis virus [38] and hepatitis C virus [39] Taken together, these results strongly suggest that the purified CSFV NS5BD24 could preferen-tially initiate either plus- or minus-strand viral RNA synthesis de novo in the absence of primers and viral or
Fig 2 RNA-dependent RNA polymerase (RdRp) activity of classical
swine fever virus (CSFV) NS5BD24 protein The RNA templates were
purified before use RNA products were separated by PAGE (8% gel
containing 7 M urea) and detected by Northern blot (A) The
228-nucleotide (+)3¢-UTR RNA transcripts were used as template (B)
RdRp assay with373-nucleotide (–)IRES RNA transcripts as
tem-plate Lane 1, absence of RNA template; lane 2, absence of NS5BD24;
lane 3, presence of NS5BD24 and RNA template; lane 4, presence of
NS5BD24GAA and RNA template The position of the input RNA
template is shown as -.
Trang 6host factors This result is contrary to the previous reports
for CSFV RdRp [11], as well as BVDV RdRp [40], another
member of the Pestivirus genus, in which the major RNA
products catalyzed by RdRp were shown to be a covalently
linked double-stranded molecule generated by a copy-back
mechanism At present it is unknown whether this
discrep-ancy is caused by the various viral enzyme preparations or
different templates used In fact, reports have shown that
the RdRp of BVDV could preferentially initiate RNA
synthesis by a de novo initiation mechanism with chemically
synthesized short RNA (21 nucleotides) as a template [41],
although a primer extension RNA product was also
observed [42] Therefore, de novo initiation of RNA
synthesis might represent the preferred mechanism used
by Pestivirus RdRps in vitro
Optimal conditions forde novo RNA synthesis using
(–)IRES as template
To optimize conditions for de novo RNA synthesis by
the NS5BD24 protein, the 3¢-end of minus-strand RNA
(–)IRES, was used as template and newly synthesized RNA
products were quantified by real-time quantitative RT–PCR
(TaqMan assay), which allowed quantification of the
starting copies of the reaction, rather than the end products,
by monitoring the increment of fluorescence released [43]
We first examined the effects of time and temperature on
RNA synthesis As shown in Fig 4A, the synthesis of RNA
products occured for at least 120 min, and the preferred
temperature for the RdRp assay was 25 C (Fig 4B) The
divalent cations Mg2+ and/or Mn2+ are known to be
required for RdRp activity Our results showed that both
Mg2+ and Mn2+ supported de novo RNA synthesis;
however, Mn2+ (Fig 4D) induced RdRp activity more
efficiently than Mg2+ (Fig 4C) Maximum activity was
observed at 5–7.5 mMMn2+and 7.5–10 mM Mg2+ Th e high concentrations of Mn2+ and Mg2+ had negative impacts on RNA synthesis Next, we determined the optimal concentrations of template and protein for RdRp assay As shown in Fig 4E, the synthesis of RNA products increased withincreasing amounts of enzyme to 100 ng, excess amounts of enzyme inhibited the RdRp reaction However, a high concentration of the RNA template had
no significant inhibition on RNA synthesis (Fig 4F), which differed from the previous report for hepatitis C virus RdRp using homologous RNA as template [44] This might be
a result of the various enzymes and template used in the RdRp reaction
Analysis of initiation ofde novo RNA synthesis
To examine the effect of preincubation with 0.5 mMNTP
on the initiation of RNA synthesis, the RdRp mixtures were first preincubated with0.5 mM of eachNTP for 30 min,
th en furth er incubated for 90 min with 0.25 mM NTP as substrates Higher activities of de novo RNA synthesis were obtained when either (+)3¢-UTR or (–)IRES template was preincubated with0.5 mMNTP, respectively, compared to the RdRp reaction without preincubation (Fig 5A,B) When (+)3¢-UTR was used as template, preincubation with0.5 mM GTP and ATP resulted in higher activities, whereas higher activities were observed following preincu-bation with0.5 mM GTP and UTP using (–)IRES as template
Previous studies have shown that a 3¢-cytidylate in the template is preferred, by several viral RdRps, for de novo initiation of RNA synthesis [41,45] To determine whether this is also the case for CSFV RdRp, we investigated the influence of substitution of the 3¢-terminal cytidylate with guanidylate, adenylate or uridylate, on de novo RNA
Fig 3 De novo initiation of viral RNA synthesis by classical swine fever virus (CSFV) RNA-dependent RNA polymerase (RdRp) Bothviral plus- and minus-strand RNA templates were treated withsodium periodate to block the 3¢-OH group and then used as template for RdRp assay (A) Northern blot assay with (+)3¢-UTR and (–)IRES as templates Lane 1, RdRp assay withNS5BD24GAA as a control; lane 2, RdRp assay with NS5BD24 and 3¢-blocked RNA template (B) Synthesized RNA was subjected to RT-PCR RT was performed using a primer complementary to the newly synthesized minus-strand (lanes 2 and 3) or plus-strand RNA (lanes 4 and 5), followed by PCR amplification Lane 1, 100 bp DNA ladder; lanes 2 and 4, RNA template (T) used as a control; lanes 3 and 5, RT–PCR results of newly synthesized products (P); lane 6, NS5BD24 protein as a control PCR products were electrophoresed through an agarose gel and visualized by ethidium bromide staining The expected fragments were 228 nucleotides (newly synthesized minus-strand RNA) and 373 nucleotides (synthesized plus-strand RNA) in length.
Trang 7synthesis As shown in Fig 5C,D, changing the 3¢ terminal
C, of bothplus-strand and minus-strand RNA templates, to
G, A or U dramatically decreased RNA synthesis,
indica-ting that a 3¢-terminal cytidylate was necessary for efficient
de novoRNA synthesis Surprisingly, RNA polymerization
was slightly higher when the 3¢-terminal C was replaced with
U, rather than A or G, with (+)3¢-UTR as template (Fig 5C) For (–)IRES as template, RNA synthesis was slightly higher when the 3¢-terminal C was replaced withG, rather than with A or U (Fig 5D) This result was in
Fig 4 Effects of reaction conditions on
de novo RNA synthesis with (–)IRES as tem-plate The synthesized RNA was quantified by real-time RT–PCR Effects of: (A) time, (B) temperature, (C) Mg 2+ concentration, (D)
Mn2+concentration, (E) enzyme concentra-tion and (F) template concentraconcentra-tion.
Fig 5 Analysis of initiation of de novo RNA synthesis (A) and (B) The RNA-dependent RNA polymerase (RdRp) mixtures were first preincubated with0.5 m M of eachNTP for 30 min, then further incubated for 90 min with0.25 m M of eachNTP as substrates (A) (+)3¢-UTR as template (B) (–)IRES as template Lane 1, no preincubation control; lane 2, preincubation with0.5 m M GTP; lane 3, preincubation with0.5 m M CTP; lane 4, preincubation with0.5 m M ATP; lane 5, preincubation with0.5 m M UTP (C) and (D) Effect of substitution of the 3¢-terminal cytidylate with guanidylate, adenylate or uridylate on de novo RNA synthesis (C) (+)3¢-UTR as template (D) (–)IRES as template Lane 1, normal template as control; lane 2, substitution of the 3¢ C withG; lane 3, substitution of the 3¢ C withU; lane 4, substitution of the 3¢ C withA The relative activity compared with the control is shown below each lane as a percentage.
Trang 8agreement withthose of Reigadas et al [45] for h epatitis C
virus using (–)IRES as template, but differed from those of
Kao et al [41] for BVDV with synthesized short RNA as
template, who showed that changing the 3¢-terminal C to G
did not direct any product synthesis This indicated that
sequences and/or structures present in other parts of the
template might play an important role for efficient de novo
RNA synthesis by CSFV RdRp
Specific interaction between CSFV RdRp and viral RNA
templates
Viral RNA synthesis requires the initiation, recognition
and specific binding between RdRp and template RNA
The 3¢-end of several positive strand RNA viruses is
known to specifically bind to their respective RdRps
[46–50] To determine whether purified and refolded
CSFV RdRp could specifically interact with either the
plus- or minus-strand viral RNA template, North-Western
blot assays were performed tRNA was included in the
binding reaction mixtures to avoid any nonspecific
bind-ing Figure 6A shows that CSFV RdRp was able to
interact withthe 3¢-end of viral minus-strand RNA
template No detectable complexes were observed when
BSA was used as a control The specificity of the RNA–
protein interaction was confirmed by template competition
assays in which unlabelled homologous or heterologous
RNAs were preincubated withthe protein for 10 min
prior to addition of the DIG-labeled RNA template The
binding to either plus- or minus-strand RNA template
showed a concentration response, because the band
intensity decreased as the amount of unlabelled
homolog-ous RNA (cold RNA) was increased The addition of a
50-fold molar excess of unlabelled RNA almost abolished
the interaction of protein with DIG-labeled RNA
tem-plate (Fig 6B,C, lane 5) However, the unlabelled
hetero-logous RNA, like yeast tRNA, showed no reduction in
the intensity of the RNA–protein complexes, even at concentrations as high as 50-fold molar excess (Fig 6B, lane 6) These results showed that the CSFV RdRp was able to specifically interact withthe 3¢ end of bothplus-and minus-strbothplus-and viral RNA templates, bothplus-and that the C terminus of NS5B was not necessary for binding activity
At present, we do not know whether other viral or cellular proteins might interact with RNA templates or RdRp to form a replication complex for initiating CSFV RNA synthesis In fact, the binding of cellular proteins to the 3¢-end of RNA templates has been described for some viruses [51,52] Interaction of the viral proteins NS3 and NS5 has been reported in Japanese encephalitis virus [53], Dengue virus [54] and hepatitis C virus [55,56] Nevertheless, our recombinant NS5BD24 expressed in E coli has several properties that resemble the functional CSFV RdRp and permit an in vitro replication system (a) it possesses RNA polymerase activity witheither plus- or minus-strand RNA
as template, (b) it contains RNA binding activity and (c) it can initiate de novo RNA synthesis from viral RNA templates and does not require an exogenous primer This
in vitroreplication system represents a starting point in the searchfor cis-elements at the 3¢ end of RNA templates and possibly viral or cell proteins required for the initiation of viral RNA synthesis
Acknowledgements
This work was supported by National Basic Research Developmental Projects (G1999011900) and National Natural Science Foundation of China (30170214).
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Fig 6 Specific interaction between classical swine fever virus (CSFV) RNA-dependent RNA polymerase (RdRp) and viral RNA templates by North-Western blot assays Proteins were transferred to nitrocellulose membranes and renatured as described in the Materials and methods (A) Binding activity of NS5BD24 with(–)IRES RNA template Lane 1, purified NS5BD24 protein; lane 2, BSA control Specificity of NS5BD24 interaction with (–)IRES RNA template (B) and (+)3¢-UTR template (C) Template competition assays were performed withhomologous and heterologous RNAs (cold RNA) as competitors Increasing amounts of unlabeled RNA were preincubated withNS5BD24 for 10 min prior to the addition of DIG-labeled RNA template The diagram was processed using Adobe PHOTOSHOP Lane 1, no competitor; lanes 2–5, competition withfivefold, 10-fold, 20-fold and 50-fold molar excesses of unlabeled homologous RNA, respectively; lane 6, competition with 50-fold heterlogous yeast tRNA.
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