- Research article
- Open Access
Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase
© Kim et al; licensee BioMed Central Ltd. 2007
Received: 22 January 2007
Accepted: 11 July 2007
Published: 11 July 2007
Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.
To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template.
As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.
Japanese encephalitis virus (JEV) is the most common cause of epidemic viral encephalitis worldwide, with approximately 50,000 cases and 15,000 deaths annually throughout a wide geographical range . Since the prototype Nakayama strain of JEV was first isolated in 1935, epidemics and sporadic cases of Japanese encephalitis have occurred in temperate and tropical zones of Asia as well as in non-Asian regions, including Cambodia, China, Indonesia, India, Japan, Malaysia, Myanmar, Nepar, Sri Lanka, Thailand, Vietnam, the south eastern Russian federation, and Australia [2, 3].
JEV is a member of Flaviviridae family, which consists of the genera Flavivirus (JEV, dengue virus [DEN], yellow fever virus [YF], West Nile virus [WNV], Kunjin virus [KUN], Murray Valley encephalitis virus), Pestivirus (bovine viral diarrhea virus [BVDV], Classical swine fever virus [CSFV]), and Hepacivirus (hepatitis C virus, [HCV]). JEV is an enveloped, positive-stranded RNA virus whose genome consists of a single-stranded RNA molecule of approximately 11 kb. The RNA genome of JEV consists of 98-nucleotide (nt) long 5' untranslated region (UTR) with the type-1 cap structure at its 5' terminus, a single open reading frame (ORF), and a 585-nt long 3'-UTR with no poly(A) tail at its 3' terminus . The single large ORF encodes a polyprotein of ~3,400 amino acids that is subsequently processed by both host and viral proteases into three structural proteins and seven nonstructural proteins . The structural proteins (capsid, membrane, and envelope proteins) are contained in the N-terminal third of the polyprotein, while the nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are located in the C-terminal two-thirds of the polyprotein. NS5 is the largest nonstructural protein of JEV. Analysis of the amino acid sequence of NS5 led to the prediction that it carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. The 5' RNA methyltransferase activity should be located on the N-terminal portion because of the presence of conserved motifs found in other viral methyltransferases [5–7]. The C-terminal region of NS5, which contains the conserved RdRp motifs [8, 9] and the Gly-Asp-Asp (GDD) motif found in the active site of many viral RdRps , is likely responsible for the RNA polymerase activity.
RNA synthesis by various RNA virus RdRps has been shown to occur by either a de novo initiation mechanism in which first nucleotide serves as a primer to provide the 3'-hydroxyl group, or via a primer-(an oligonucleotide, a protein linked to nucleotides, or intramolecular self priming) dependent mechanism [11–16]. De novo initiation results in the synthesis of various RdRp products including a template-size product initiated from the most 3'-end or an RNA molecule smaller than the template (via internal initiation) [11, 15, 17]. Although de novo initiation has been demonstrated for a number of Flaviviridae RdRps, including those from WNV, DEN, KUN, BVDV, CSFV, and HCV [15, 17–22], the biochemical properties JEV RdRp and its RNA synthesis initiation mechanism have not been yet analyzed in detail due to the lack of an in vitro RdRp assay system. In this work, we expressed and purified JEV NS5 protein from Escherichia coli and characterized its biochemical properties and in vitro RNA synthesis initiation mechanism.
Expression and purification of recombinant JEV NS5 protein
Analysis of the RdRp activity of JEV NS5
De novo initiation of RNA synthesis from the plus- and minus-strand 3'-UTR of JEV genome
De novo internal initiation of RNA synthesis from the 83-nt RNA template
TNTase activity of JEV NS5 protein
In this study, we established an in vitro JEV RdRp assay system with a functional full-length JEV NS5 protein and characterized the mechanism of initiation of RNA synthesis. Results from this study showed that the recombinant JEV NS5 exhibits both RdRp and TNTase activities. The enzyme was capable of synthesizing RNA by a primer-dependent manner with a homopolymeric RNA template poly(A) and by de novo initiation with the RNA templates derived from JEV genome. Several reports showed that the TNTase activity is a common intrinsic property of many RdRps of Flaviviridae. The recombinant RdRps of HCV and BVDV have been shown to possess a TNTase activity under certain reaction conditions [24, 25]. CSFV NS5B protein also was shown to possess TNTase activity, which added a single nucleotide to the 3' end of the 3'-UTR RNA template . The JEV NS5 exhibited a weak but detectable level of intrinsic TNTase activity only when substrate ribonucleotides were limited (Fig. 9, lanes 3 and 4). Because the TNTase activity was detected only when limiting amount of cold UTP was added to the reaction mixture, its in vivo function, if any, remains unclear.
The JEV NS5 has a strong preference for Mn2+ over Mg2+ ion for RNA synthesis (Figs. 3 and 4). Considering that the intracellular concentration of Mn2+ is much lower than Mg2+ , the stringent Mn2+-dependent RdRp activity of JEV NS5 may not allow efficient JEV replication in vivo. Alternatively, this Mn2+-dependent RdRp activity might be simply an artifact of the unnatural in vitro conditions. It is also possible that this might be due to conformational difference of the recombinant JEV NS5 from the one in the virus-infected cells, in which JEV NS5 was likely to be membrane associated or to form a complex with other viral and/or cellular proteins. Nevertheless, similar stringent Mn2+-denpedent RNA synthesis using the homopolymeric RNA template poly(C) has been demonstrated with recombinant RdRps DEN and WNV . Furthermore, when a DEN minigenome consisting of the 5'-end RNA (224 nt) and the 3'-end RNA (492 nt) of the viral genome was used as a template, the DEN RdRp activity was supported either by Mg2+ or Mn2+, with the latter cation being a better cofactor. Our result is however contradictory to that obtained previously by in vitro RdRp assay using the JEV replication complex (RC) isolated from infected cells . The RC displayed a strict dependence on Mg2+ with absolutely no RdRp products being synthesized from the endogenous viral RNAs associated with the RC in the absence of Mg2+. The requirement of Mg2+ for RNA synthesis by JEV RC might be due, in part, to its role as a cofactor for other viral and/or cellular proteins associated with the viral RNA polymerase.
During the course of flavivirus replication, minus-strand RNA is synthesized from the plus-strand genomic RNA. This intermediate form of RNA serves as a template for the synthesis of plus-strand genomic RNA that is synthesized at 10- to 100-fold higher levels than the minus-strand RNA . Results from our in vitro RdRp assays using the 3'(+)UTR and 3'(-)UTR templates also supported the notion that a greater amount of plus-strand RNA than minus-strand RNA is synthesized in JEV- and DEN-infected cells [28, 29]. The ratio of plus-to-minus strand RNA in the cells infected with these viruses was approximately 10:1. As shown in Fig. 5C, we demonstrated that the 3'(-)UTR served as a better template than the 3'(+)UTR for JEV NS5. This in vitro result also agreed with the result showing that the HCV NS5B RNA polymerase produces RdRp products more efficiently using the 3'(-)UTR than the 3'(+)UTR of the plus-strand viral RNA . Such preference for plus-strand 3'-UTR over minus-strand 3'-UTR for RNA synthesis initiation might be correlated with the binding activity of RdRps to these cis-acting RNA elements. Indeed, a recent report showed that CSFV NS5B protein, a viral RdRp, recognizes the 3'(-)UTR more efficiently than the 3' (+)UTR . Regarding the mechanism of initiation of RNA synthesis, JEV NS5 appears to be able to initiate RNA synthesis either de novo or by intramolecular priming in vitro using the viral genome-derived RNA templates tested in this work. From the 3'(+)UTR, JEVNS5 produced template size RNA as a major RdRp product, whereas bigger-than-template products were synthesized from the 3'(-)UTR RNA template (Fig. 5C). The nature of this bigger-than-template product is not clear at the present time, although it might be generated by extension of the 3'-end of the template by a snap-back priming mechanism or by extension of the newly synthesized nascent RNA products. The long single-stranded region at the 3'-end of the predicted secondary structure of the 3'(-)UTR might allow intramolecular priming.
Both nucleotide sequence and structure of 3'-UTR of plus-strand RNA viruses contain a cis-acting signal essential for the initiation of viral RNA replication. Although the size and sequence of the 3'-UTR vary among different flaviviruses, its secondary structure comprising stable stem-loops is predicted to be highly conserved. The last 80 to 90 nts at the 3'-end of various flavivirus genomes have been predicted to form stable stem-loop structures [31–33]. The last 83-nts of the 3'-UTR of JEV are predicted to form a stable stem-loop structure (Fig. 7C), which is similar to that formed with the 3'-end of the KUN genome . The ability of JEV NS5 to use the 83-nt RNA template as a minimal template allowed us to explore its role in RNA synthesis. Our results revealed that JEV NS5 initiates RNA synthesis from an internal nucleotide U81, the third nucleotide from the 3'-end of the template (Fig. 7). We also observed that, although it is a very minor one, wild-type 83-nt RNA (83 WT), 83(Δ1), and 83(+U) generated an RNA product of 83-nt (Fig. 8). Because de novo initiated products from the 3'-end of these templates would generate 82, 83, and 84-nt product from 83(Δ1), 83 WT, and 83(+U), respectively, this product appears to be the 3'-end extended nascent RNA. Internal initiation of RNA synthesis was observed similarly with other Flaviviridae RdRps. For example, the penultimate cytidine at the 3'-end of KUN plus- and minus-strand RNA was shown to be essential for KUN RNA replication . In addition, Oh et al.  and Kim et al.  showed that RNA synthesis initiates from an internal region of the 98-nt X-RNA template at the 3'-end of HCV genome, which is similar in terms of the RNA synthesis initiation mechanism to the result shown with JEV NS5 in this study. Because initiation of RNA synthesis from an internal nucleotide during viral RNA replication will result in loss of 3'-end genetic information, cellular and/or viral factors may play a role in the initiation of RNA synthesis from the 3'-end of genome. Previously, Chen et al. (8) showed that both JEV NS3 helicase and NS5 bind to the 3'-UTR of JEV genomic RNA. Moreover, the 36-kDa Mov34 protein was known to bind the 83-nucleotide 3' stem-loop structure recognized by the NS5 protein [23, 36]. Those known viral and cellular proteins interacting with the 3'-end minimal cis-acting elements as well as other unknown factors might allow for JEV NS5 protein to initiate RNA synthesis from the 3'-end. The recombinant JEV NS5 protein will permit evaluating the effect of such trans factors on initiation of RNA synthesis.
Previous studies proposed that the 5' and 3' ends of the flavivirus RNA genome are able to interact directly between the cyclization sequence within the 3'-UTR and its complementary sequence in the capsid coding region following the 5'-UTR of the viral genome . The cross-talk between these conserved cis-acting RNA elements of various flaviviruses was shown to be required for viral replication [38–40]. In vitro RdRp assays using DEN-infected cell lysates and recombinant viral RdRp from WNV showed that minus-strand RNA synthesis requires the interaction between the two terminal regions on the plus-strand viral RNA template through a cyclization motif, [18, 21, 41]. These previous results proposed that RNA synthesis from the 3'-UTR of plus-strand RNA requires the 5'-terminal region of the viral genome supplied in cis or trans so that cyclization of the 5'- and 3'-terminal regions forms a pan-handle-like structure. In contrast to in vitro RdRp assay results obtained with the above described flavivirus RdRps, our results demonstrated that JEV NS5 protein is capable of using the JEV 3'(+)UTR and the 83-nt RNA as templates (Fig. 5). RNA synthesis from these templates did not require the 5'-terminal cyclization motif in cis or trans. This result suggests that the sequence and/or structure of the 3'(+)UTR and the 83-nt RNA is sufficient for the NS5 to recognize the template and to initiate RNA synthesis, and indicates that cyclization of the JEV genome is not required for RNA synthesis in vitro. Thus, it is tempting to speculate that cyclization of the JEV RNA genome in vivo, if any, via direct RNA-RNA interaction or indirect interaction through other cellular and/or viral proteins, is involved in viral genome translation.
We established an in vitro JEV RdRp assay system with an enzymatically active recombinant JEV NS5 protein. This recombinant JEV alone was able to recognize the cis-acting elements on both plus- and minus-strand 3'-ends. Like some of other flavivirus RdRps, it carries both RdRp and intrinsic TNTase activities. Its internal do novo RNA initiation from the 83-nt RNA template suggests that JEV RNA replicase complex might require viral and/or cellular proteins to direct RNA synthesis initiation form the 3'-end in vivo. The recombinant JEV NS5 protein will facilitate the analysis of the roles of such factors in initiation of RNA synthesis. The functional NS5 protein will also be useful for the development of target-specific inhibitors of JEV replication.
Cells and virus
Baby hamster kidney cells (BHK-21) were grown at 37°C in minimum essential medium (MEM, Invitrogen Life Technologies) supplemented with 5% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin sulfate (Invitrogen). The Nakayama strain of JEV, which was prepared from virus-infected mouse brain, was provided by the Department of Viral Disease, Korea National Institute of Health and used in our study. BHK-21 cells were infected with JEV at an multiplicity of infection of five as described previously .
Construction of the recombinant JEV NS5 expression vector
To obtain a cDNA fragment encoding JEV NS5, viral genomic RNA from culture supernatants of JEV-infected BHK-21 cells was extracted with Trizol LS reagent (Invitrogen Life Technologies). After phenol/chloroform extraction, purified RNA was precipitated with isopropanol, washed once with 70% ethanol, and dissolved in RNase-free water. The RNA was reverse transcribed using Superscript II reverse transcriptase (Gibco-BRL) and the reverse primer 5'-GGGGTACCGATGACCCTGTCTTCCTG-3' as instructed by the manufacturer. The NS5 coding sequence was amplified by PCR, using Vent DNA polymerase (New England Biolabs) along with the forward primer (5'-CTAGCTAGCGGAAGGCCCGGGGGCAGG-3') and the reverse primer. The PCR product was digested with Nhe I and Kpn I and ligated into a similarly digested pTrcHisB (Invitrogen) vector to make the plasmid pTrcHisB-JEVNS5. The JEV NS5 mutant NS5D668A with a substitution of the first Asp with Ala in the GDD motif was generated by site-directed mutagenesis with two sequential rounds of PCR using oligonucleotides containing the target mutation as described previously . The presence of the desired mutation was verified by DNA sequencing.
Expression and purification of recombinant JEV NS5 protein from E. coli
JEV NS5 protein was expressed in E. coli TOP10 cells (Invitrogen) transformed with pTrcHisB-JEVNS5. The transformed cells were grown in LB medium containing 100 μg of ampicillin per ml to an optical density at 600 nm of 0.6–0.8 at 37°C, and protein expression was induced at 18°C for 12 h by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The NS5 protein was purified by metal affinity chromatography using Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen) resin as described previously . The bound JEV NS5 proteins were step-eluted with the binding buffer containing 50 to 500 mM imidazole. JEV NS5-containing fractions were collected, dialyzed against a gel filtration column buffer (50 mM Tris-HCl [pH 7.8], 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA), and then applied to a Sephacryl S-200HR column (Amersham Biosciences) at a flow rate of 0.5 ml/min. JEV NS5-containing fractions were collected and dialyzed against buffer A (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM DTT, 10% glycerol), and purified further by applying to an SP-Sepharose column (Amersham Biosciences). Adsorbed proteins were eluted with a 5 ml linear gradient of NaCl from 0.1 to 1 M in buffer A. Small aliquots of the NS5-containing fractions were stored at -80°C after dialyzing against buffer A. Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin as a standard.
RNA template preparation
The DNA templates for 3'(+)UTR (representing the 3'-UTR of JEV genome), 3'(-)UTR RNA (representing the region complementary to the 5'-UTR of JEV genome), the 83-nt RNA template (representing the 83-nt RNA from the 3'-end of JEV genome) were obtained by PCR with Vent DNA polymerase and the specific primers for 3'(+)UTR RNA (5'-TAATACGACTCACTATA GCTAGTGTGATTTAAAGTA-3' and 5'-AGATCCTGTGTTCTTCC-3'), 3'(-)UTR RNA (5'-AGAAGTTTATCTGTGTG-3' and 5'-TAATACGACTCACTATA GGTTATCTTCCGTTCTAA-3'), and the 83-nt RNA (5'-TAATACGACTCACTATA GATCTTCTGCTCTATCTC-3' and 5'-AGATCCTGTGTTCTTCC-3'). The T7 RNA polymerase promoter sequence is underlined, and the sequence complementary to the JEV genome sequence is shown in boldface and italic. pBACSP6/JVFL/Xba I , which was kindly provided by Dr. Y. M. Lee at Chungbuk National University, Cheongju, Korea, was used as a template for PCR. Various DNA templates for derivatives of the 83-nt RNA were generated by PCR using a set of specific primers. The PCR-amplified DNA products were gel purified and used for in vitro transcription using T7 RNA polymerase as described previously . RNA concentrations were estimated by measuring the absorbance at 260 nm.
Enzymatic assays and analysis of products
RdRp assays were performed with 500 ng of purified JEV NS5 RdRp in a total volume of 25 μl containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 25 mM potassium glutamate, 1 mM MgCl2, 2.5 mM MnCl2, 1 mM DTT, 10% glycerol, 20 units of RNase inhibitor (Promega), cold ribonucleotide mixture (0.5 mM each ATP, CTP, and GTP, and 5 μM UTP), and 10 μCi of [α-32P] UTP (3,000 Ci/mmol; Amersham Biosciences). The reaction mixture was incubated with 200 ng of RNA template for 2 hr at 30°C. For RdRp reaction with poly(A) template, 1 μg of RNA was added to the reaction mixture as described above, except that 10 μM UTP was included in the reaction with 2 μCi of [α-32P] UTP. The RdRp reactions were performed in the absence or presence of 10 pmol of oligonucleotide (U)20. For the TNTase assay, 10 μCi of [α-32P] UTP mixed with or without 10 μM of cold UTP was used as a single ribonucleotide in the RdRp reaction mixture with the 83-nt RNA as a template. [γ-32P]-ATP incorporation assay was performed as described above for the standard RdRp assay, except that the reaction mixture contained cold ribonucleotide mixture (0.5 μM each CTP, GTP, and UTP, and 1 μM or 5 μM ATP) and 0.33 μM [γ-32P]-ATP (6,000 Ci/mmol, 10 mCi/ml; Amersham Biosciences).
RdRp reaction products were precipitated and resuspended in a denaturing loading buffer as described previously . After heat denaturation and quick chilling on ice, the RNA products were resolved on 8 M urea-5% polyacrylamide gels (20 × 20 cm), unless otherwise specified. For size mapping, RNA products were separated in 8 M urea-6% denaturing polyacrylamide gels (20 × 45 cm). The gels were stained with ethidium bromide, photographed to locate the template positions, and then dried. The dried gels were exposed to X-ray film (BioMax XAR, Kodak) for autoradiography. The amounts of 32P-UMP incorporated into the products were measured in a LS-6500 multi-purpose scintillation counter (Beckman).
Nuclease S1 treatment
RdRp products were digested with 4 units of nuclease S1 per ml (Promega) at 37°C for 30 min in nuclease S1 digestion buffer (30 mM NaOAc [pH 4.6], 1 mM ZnSO4, 5% glycerol) containing either a low (50 mM) or high (500 mM) concentration of NaCl.
Preparation of RNA size markers
For 5'-end 32P-labeling of the 83-nt RNA, in vitro transcripts were dephosphorylated with calf intestine alkaline phosphatase (Takara Bio Inc.), and then phosphorylated with T4 polynucleotide kinase (New England Biolabs) in a final reaction volume of 50 μl containing 50 μCi [γ-32P] ATP and 0.1 mM cold ATP. After incubation at 37°C for 30 min, free nucleotides were removed using a Sephadex G-25 spin column, and the labeled RNA was purified from the denaturing polyacrylamide gel as described previously . The 32P-labeled 83-nt RNA was partially digested by incubation in Na2CO3/NaHCO3 buffer (pH 10) for 10 min at 90°C.
Prediction of RNA secondary structure
SDS-PAGE, Western blot analysis, and MALDI-TOF analysis
For Western blot analysis, protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Amersham Biosciences). The samples were blocked using 5% skim milk in TBST (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20), then reacted with an anti-His antibody (Qiagen) at a 1:1,000 dilution, followed by incubation of goat anti-mouse lgG conjugated with peroxidase (Sigma-Aldrich). The immunoblots were washed and then immunoreactive protein bands were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences). Silver staining of gels and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis were performed as described previously .
This work was supported by the Basic Research Program of the Korea Science and Engineering Foundation (RO1-2004-000-10382-0) and by a Korea Research Foundation Grant (KRF-2004-C00148) funded by the Korean Government. JSY, CMK, and JHK were supported in part by the BK21 Program of the Korea Ministry of Education and Human Resources Development.
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