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Originally published In Press as doi:10.1074/jbc.M501393200 on May 24, 2005

J. Biol. Chem., Vol. 280, Issue 29, 27412-27419, July 22, 2005
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Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5'-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase*

Changsuek Yon{ddagger}§, Tadahisa Teramoto{ddagger}§, Niklaus Mueller{ddagger}, Jessica Phelan{ddagger}, Vannakambadi K. Ganesh¶, Krishna H. M. Murthy¶, and R. Padmanabhan{ddagger}||

From the {ddagger}Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington D. C. 20057 and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, February 7, 2005 , and in revised form, May 5, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mosquito-borne Flavivirus genus, in the Flaviviridae family, includes human pathogens of global distribution and prevalence (for reviews, see Refs. 1-3), and Dengue viruses (DEN)1 types 1-4 cause the most common infection encountered in humans (4). The diseases caused by DEN infections include from dengue fever, usually a self-limiting disease, to more severe forms, dengue hemorrhagic fever and dengue shock syndrome. These diseases pose a significant threat to humans living in DEN-infected Aedes aegypti mosquitoes endemic in areas that inhabit two-thirds of world population (5)

DEN genome is a single-stranded RNA (10,723 nt in length for DEN2 New Guinea C strain used in this study (6)) of positive polarity. The viral RNA contains a long open reading frame coding for a polyprotein precursor. The polyprotein is processed into mature structural proteins, capsid (C), precursor membrane (prM), and envelope (E) and at least seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, by cellular signal peptidase and viral serine protease in the endoplasmic reticulum (for review, see Ref. 7). The 5'-end of the viral RNA is modified by a type I cap structure (m7GpppN; 2'-OH moiety of N is methylated).

DEN2 NS3 is a multifunctional protein of about 69 kDa. It includes a serine catalytic triad within the N-terminal 185 amino acid residues. The protease domain is activated by the hydrophobic protein NS2B which serves as a cofactor for the protease and forms a complex in the infected cells (8-12). NS2B has three hydrophobic regions flanking a conserved hydrophilic domain. The hydrophilic domain of NS2B (NS2BH) alone is sufficient for protease activity in processing of the 2B/3 site in the precursor expressed in transfected cells or in an in vitro protease assay (13, 14). NS2B is an endoplasmic reticulum resident integral membrane protein (14).

The region C-terminal to the protease domain of NS3 has conserved domains found in the DEXH family of NTPases/RNA helicases (15). The NTPase activity of NS3, involved in the hydrolysis of the {gamma}{downarrow}{beta}{alpha} phosphoric anhydride bond (shown by an arrow) of NTP has been reported for several flaviviruses including DEN2 (16-20). In addition, NS3 has the 5'-RNA triphosphatase activity (5'-RTPase), capable of hydrolyzing the {gamma}{downarrow}{beta}{alpha} phosphoric anhydride bond of triphosphorylated RNA as shown for an N-terminal-truncated fragment of West Nile virus NS3 partially purified from infected cells (21) or full-length NS3 expressed and purified from Escherichia coli (20, 22). The 5'-RTPase is the first of the three sequential enzymatic reactions that are involved in the addition of 5'-cap to RNA (for review, see Ref. 23). Based on mutation analysis and competition experiments using ATP substrate and analogs, it was concluded that the RNA-stimulated NTPase activity and the 5'-RNA triphosphatase activity involve a common active site determinant(s) (20, 22).

The ATPase activity is intrinsic to viral and cellular RNA helicases and is required for unwinding of a double-stranded RNA substrate (for reviews, see Refs. 24 and 25). RNA helicase activities of NS3 of hepatitis C virus, a member of Hepacivirus genus (26-35), and the p80 of bovine virus diarrhea virus, a member of the Pestivirus genus (36, 37), have been well characterized. However, the RNA helicases of mosquito-borne flaviviruses have not been well studied. The N-terminal-truncated DEN2 (16, 20) and Japanese encephalitis virus NS3 (38) were expressed in E. coli and purified to demonstrate the RNA helicase activity. The RNA helicase activity of the full-length NS3, its RNA binding properties, the kinetic parameters or the influence of the protease cofactor, NS2BH domain, on the RNA helicase activity has not been previously studied to date. In this study we report that the full-length NS3 with or without NS2B cofactor domain, expressed in E. coli and purified, exhibits a catalytically more efficient RNA helicase activity than the N-terminal-truncated NS3 helicase domain, suggesting that the protease domain enhances RNA helicase activity. A functionally active serine catalytic triad is not required for this enhancement of RNA helicase activity as shown by mutagenesis of the protease catalytic triad residue, H51A.

The multifunctional NS3 protein exists in a complex with NS5, which itself has two enzyme activities, the 5'-RNA O- methyl transferase involved in 5'-capping and the RNA-dependent RNA polymerase required for viral RNA replication in DEN2-infected cells (39) (for review, see Ref. 40). Therefore, we examined the role of NS5 interaction on the NTPase/RNA helicase and the RTPase activity of NS3. Our results show that the NS5 stimulates the NTPase activity of NS3 in a dose-dependent manner until an equimolar stoichiometry is attained, which supports the notion that the heterodimeric NS3·NS5 complex is the functional unit involved in unwinding double-stranded RNA during replication. Furthermore, RNA binding analyses by gel shift assays show that NS3 binds to RNA, and a positively charged motif, RKRK, which is conserved in Flavivirus NS3, is required for this RNA binding activity. Mutation of this motif significantly reduced the NTPase/RNA helicase activity of NS3, suggesting that this motif is an important determinant in NTPase/RNA helicase function of NS3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—The construction of the NS3FL expression plasmid with a His6 tag at the N terminus is described previously (22). NS2BH(QR)-NS3FL expression plasmid, which contains the hydrophilic domain of NS2B shown to be sufficient for activation of the NS3 protease domain (13, 14), was constructed as follows. The PCR primers used were 5'-CGCGGATCCGCCGATTTGGAACTGGAGAGAGCCGCC-3' (the BamHI site is underlined; from the 5'-TCC (Ser) codon to GCC (Ala) codon represents amino acid positions 48-57 of NS2B as described previously (14)) and 5'-TTGGCGCGCTGTTCTTCCTCTTCGTTTTTTATCGAC-3' (the BssHII site is underlined; the primer represents amino acid positions 93-85 of NS2B); the former codes for RGSADLELERAA57, and the latter codes for 85SIKNEEEEQ93. The primers used for NS3-pro were 5'-TTGGCGCGCTGGAGTATTGTGGGATGTCCCTTCACC-3' (representing the sequence coding from the CGC and GCT, Arg and Ala codons, respectively (130R{downarrow}AGV, the arrow denotes a protease-sensitive site at the N terminus of NS3 with the underlined BssHII site) and the reverse primer, 5'-CCCAAGCTTACTTTCGAAAAATGTCATCTTCGATCTnt5049-3' (the HindIII site is underlined). The products of these two PCR reactions were cloned into the TA vector (Invitrogen). The positive clones were identified, and the plasmid DNAs were digested with either BamHI + BssHII or BssHII + HindIII. Both fragments were cloned into the pQE30 vector (Qiagen) at BamHI and HindIII sites, giving rise to pQE30-NS2BH(QR)-NS3pro. To generate the NS2BH(QR)-NS3FL, the NS3-pro region in the pQE30-NS2BH(QR)-NS3pro was replaced by NS3FL as follows. pQE30-NS3FL (22) and pQE30-NS2BH(QR)-NS3pro were both digested with NdeI. After removing the NS3pro fragment, the corresponding fragment of NS3FL was inserted into the digested pQE30-NS2BH, generating pQE30-NS2BH(QR)-NS3FL. To generate the pQE30-NS2BHQR-NS3FL(H51A) plasmid, the His51 -> Ala mutation of the serine protease catalytic triad was inserted by replacing the wild type BamHI-BssHII fragment with the corresponding fragment from the mutant NS2BH(QR)-NS3-pro plasmid described previously (14). The mutant pQE30-NS3FL (184RKRK -> QNGN; hereafter referred as 4M) plasmid was constructed by overlap PCR (41, 42) as follows. Four primers were synthesized: Primer 1, 5'-CTAATGCATAAAGGAAAGAGGATT-3' (nt 4693-4716); Primer 2, 5'-CAAAACGGAAATTTGACCATCATGGACCTCCAC-3' (the 4-amino acid region mutated spans 5071-5082, and the primer spans up to position 5103); Primer 3, ATTTCCGTTTTGAAAAATGTCATCTTCGATCTC-3' (reverse primer, nt positions 5050-5082; underlined in 2 and 3 is the 4M mutant region); Primer 4, 5'-CAGGATTAATGTCCTCAGGCCCCG-3' (nt 5161-5184). Individual PCR reactions were carried out using primers 1 + 3 and 2 + 4, PCR products were mixed, and a third PCR was done using primers 1 and 4 as described (42). The PCR products were cloned into pGEM-T-Easy Cloning vector (Promega, Madison WI). All constructs were verified by sequencing. The PCR product (491 bp) containing the 4M mutation was cloned into pQE30 vector (Qiagen) containing the wild type NS3FL cDNA (22) between the NsiI (nt 4696) and Bsu36I (nt5166) sites within the NS3 coding sequence (see Fig. 1).

Purification of Recombinant Proteins—E. coli strain, Top 10F' (Invitrogen), or BL21 was transformed with appropriate expression plasmid, and the cells were grown at 37 °C in LB medium containing ampicillin (100 µg/ml) and 0.5% w/v glucose to an optical density of 0.6 at 600 nm. Cells were centrifuged at 6000 x g and resuspended in glucose-free LB+ ampicillin (100 µg/ml), and isopropyl-1-thio-{beta}-D-galactopyranoside (Sigma) was added to a final concentration of 1 mM. Cells were grown for 5 h at 30 °C, harvested by centrifugation, and stored at -80 °C until use. For purification recombinant proteins, the bacterial pellets were resuspended in native lysis buffer containing 100 mM sodium phosphate buffer, pH 7.5, 300 mM NaCl, and 40% glycerol. Cell lysates were loaded onto a cobalt-based immobilized metal affinity column (TalonTM, Clontech). After incubation and then washing, the recombinant proteins were eluted with 500 mM imidazole. The fractions containing the highest protein concentration were pooled, dialyzed against a buffer containing 50 mM sodium phosphate, pH 7.5, 300 mM NaCl, and 40% glycerol. In some cases the recombinant proteins were purified by using the fast protein liquid chromatography HiTrap Chelating HP 1-ml column) (AKTAprime system from Amersham Biosciences). The proteins were eluted with 20 mM Tris-Cl, pH 7.4, 500 mM NaCl, and 500 mM imidazole. Peak fractions were pooled and loaded again to the HiTrap SP FF (1 ml) column. The fractions with the highest protein concentrations were pooled and dialyzed against 20 mM Tris-HCl, pH 7.5, 75 mM NaCl, and 40% glycerol.

RNA Substrates—For the electrophoretic mobility shift assays and the 5'-RTPase assays, RNA of 100 nucleotides in length containing sequences from the 5'-end of DEN2 RNA (which includes the 5'-untranslated region of 96 nucleotides) was obtained by in vitro transcription of the PCR product. The template for PCR was the pSY2 plasmid (encoding the subgenomic RNA (43)). The concentration of the RNA was measured by using a spectrophotometer, and the integrity of RNA was verified by partially denaturing PAGE (3.5% containing 7 M urea) followed by ethidium bromide staining. For RNA helicase assays two oligomer RNAs, a 30- and 15-mer (44), were synthesized (Dharmacon). The sequences of the oligomers are 5'-CAUCAUGCAGGACAGUCGGAUCGCAGUCAG-3' and 5'-GUAGUACGUCCUGUC-3. The 30-mer was labeled at the 5'-end using T4 polynucleotide kinase at 37 °C for 2 h. Then the labeled oligomer was extracted by phenol/chloroform followed by ethanol precipitation. Unincorporated [{gamma}-32P]ATP was further removed by passing through two Sephadex G-25 spin columns (Bio-Rad). The purified radiolabeled oligomer was mixed with the complementary strand, heated to 95 °C for 10 min, and cooled slowly to room temperature (>3 h).

NTPase and RTPase Assays—The NTPase and RTPase assays were based on the original colorimetric method described several decades ago to quantify the inorganic phosphate present in serum (45, 46) and more recently in a study of HCV NS3-associated NTPase (47). The method used in our study has slight modifications. Briefly, the reaction mixture (50 µl) consisted of 25 mM HEPES-K+, pH 7.5, 1 mM MgCl2, 1 mM NTP (unless the NTP concentration was varied), and 140 nM purified NS3 protein. For RTPase assays, the NTP was substituted with triphosphorylated RNA (100 nucleotides in length). The reaction mixtures were incubated for 30-60 min (unless otherwise indicated) at 37 °C. Reactions were stopped by adding 10 µl of trichloroacetic acid. A freshly prepared solution of 140 µl containing a 6:1 ratio of 0.42% ammonium molybdate in 1 N H2SO4 and 10% ascorbic acid was added and incubated for 20-40 min at 42 °C. The "molybdenum blue" formed was measured at A780 nm using a spectrophotometer (SpectraMax, Molecular Devices).

RNA Helicase Assay—The reaction mixture (20 µl) for RNA helicase consisted of 20 mM HEPES-KOH, pH 7.0, 2 mM dithiothreitol, 1.5 mM MgCl2, 2.5 mM ATP, 500 nM purified protein (unless the protein amounts were varied), and 5 nM substrate. The reaction was carried out at 37 °C for various times and then terminated by adding 5 µl of 5x RNA loading dye (100 mM EDTA and 0.7% SDS). Samples were loaded onto a 12% native polyacrylamide gel. The gel was electrophoresed at 100 V for 1.5 h, dried, and subjected to autoradiography. The ratio of released single strands versus the total of single and double strands was quantified by PhosphorImager (Molecular Dynamics).

Electrophoretic Mobility Shift Assay—The reaction mixture (20 µl) contained 20 mM Tris-Cl, pH 7.5, 75 mM NaCl, 10 mM MgCl2, 1% Ficoll 400, 20-150 nM 32P-labeled DEN2 RNA, 0.5-1 µg of NS3FL or NS3FL(4M). The reaction mixtures were incubated for 30 min on ice, and 3 µl of loading buffer (40% sucrose) was added. The reaction mixtures were developed using 1.5% acrylamide, 1% agarose composite gel. The bands were identified by autoradiography after drying the gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and purification of DEN2 NS3FL proteins. A previous study from our laboratory indicated that the N-terminal-truncated DEN2 NS3 (NS3del.2, referred to as NS3{Delta}160) protein had basal NTPase that was stimulated by the addition of poly(A). NS3{Delta}160 had the deletion of the serine protease domain but contained all the conserved domains attributed to NTPase/RNA helicases of DEXH family members. The kinetic constants of the NTPase activity of NS3{Delta}160 were close to those of other flaviviral NTPases. However, the RNA helicase activity of NS3del.2 required 2.7 µM concentrations of purified NS3del.2 protein to unwind less than 5% of a 29-bp RNA duplex. On the other hand, the hepatitis C virus NS3 and the bovine diarrhea virus p80 RNA helicases required 0.1-1 pmol (~10-100 nM) of enzyme for unwinding similar substrates (27, 36). We also reported that the RNA-stimulated NTPase activity of NS3del.2 was abolished by mutation of the positively charged motif, 184RKRK -> 184QNGN, of NS3 or by deletion of an additional 20-amino acid residues from NS3{Delta}160 (NS3{Delta}180) even though the latter mutant still contained the 184RKRK motif very close to the N terminus. In either of these two mutants the basal NTPase activity was still retained (16). From these results we concluded that the 184RKRK motif, although not required for basal NTPase activity, played an important role in the RNA-mediated stimulation of NTPase. The mechanism for requirement of the positively charged motif in RNA-stimulated NTPase was unknown. In this study we hypothesized that the weak RNA helicase activity associated with the NS3{Delta}160 protein might be because of suboptimal folding of the NS3{Delta}160 protein and/or binding to double-stranded RNA substrate in contrast to the RNA helicases associated with the full-length NS3 of hepatitis C virus or the p80 of bovine viral diarrhea virus. We also considered the alternate possibility that the DEN2 RNA helicase may be stimulated by interaction with other viral components such as NS5, the RNA-dependent RNA polymerase, with which NS3 forms a stable complex in Flavivirus-infected cells (39, 48). Moreover, none of the mosquito-borne flaviviral NS3 RNA helicases have been studied in detail to date.

In this study we launched detailed biochemical and kinetic analyses of NTPase/RNA helicase activities of full-length NS3 (NS3FL) containing both the serine protease domain at the N terminus and the conserved NTPase/RNA helicase domains at the C terminus. Because the protease domain of NS3 interacts with the hydrophilic domain of NS2B (NS2BH), we sought to examine the effect of this interaction on NTPase and RNA helicase activities. To this end we also constructed the expression plasmid, NS2BH(QR)-NS3FL, encoding the precursor protein in which the NS2BH is linked to the protease domain of NS3 flanking the two amino acid residues, QR, occupying the P2 and P1 positions of the protease cleavage site. The recombinant protein expressed from this plasmid underwent cis cleavage at the protease-sensitive site QR{downarrow} due to interaction of NS2BH with the NS3 protease domain to produce the non-covalent binary complex, NS2BH/NS3FL. We also constructed the NS2BH(QR)-NS3FL(H51A) expression plasmid containing the mutant catalytic triad residue (H51A). The protein expressed from this plasmid did not undergo cis cleavage and was in the precursor form (see Fig. 2A) as expected. To analyze the role of 184RKRK motif in NTPase and RNA helicase activities of NS3FL, the NS3FL containing the mutant motif 184QNGN was constructed (Fig. 1). The transformed E. coli TOP10F' cells were grown, and the proteins were expressed and purified as described under "Materials and Methods." Fig. 2 shows the Coomassie Blue-stained SDS-PAGE of the purified proteins. To study the effect of NS5 on the NTPase and RNA helicase activities of NS3, NS5 protein was expressed from the pMHA77-3 plasmid as described previously (49) (Fig. 2C). The NS2BH(QR)-NS3FL precursor underwent partial cleavage at the QR{downarrow} site during purification from the TalonTM column as shown in Fig. 2D (lane 2). We observed that if the pH of the buffer used for dialysis of the protein was at 9.0, the cis cleavage of the QR{downarrow} site was enhanced and was essentially complete (Fig. 2D, lane 1). The identities of the purified proteins were confirmed by immunoblotting using either anti-NS2B (Fig. 2E) or anti-NS3 antibodies (Fig. 2F; data not shown). The NS3FL and NS3FL(4M) proteins were expressed and purified as described under "Materials and Methods" (Fig. 2B; data not shown).



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  		FIG. 1.
Plasmid constructs. The construction of NS3FL was described previously (22). The construction of the other three expression plasmids are described under "Materials and Methods."

 
NTPase Activities of NS3FL, NS2BH(QR)/NS3FL, and NS2BH(QR)-NS3FL (H51A) Proteins—In our previous study of the biochemical characterization of the NTPase activity of the NS3{Delta}160 protein, a coupled system that measured the oxidation of NADH at 340 nm (16, 18, 50) was employed to assay the NTPase activity. In this study we used an assay adapted to microtiter plate format that is based on the conversion of the Pi formed by ATP hydrolysis to intense blue color in the presence of ammonium molybdate and ascorbic acid (45, 46) with slight modifications as described under "Materials and Methods." Under these conditions the colorimetric reaction obeyed Beer's law for at least up to 100 µM Pi. Pi was the preferred substrate over PPi, and the rate of the NTP hydrolysis was linear with increasing concentrations of the enzyme (data not shown). The NTPase activity of NS3 at various ATP concentrations exhibited Michaelis-Menten kinetics (Fig. 3). The kinetic parameters of the NS3FL proteins were then determined from the Lineweaver-Burk plots (Fig. 3, inset). The results from several assays indicated that the apparent Km of NS3FL for ATP was 191 µM, the apparent Vmax was 6.05 ± 0.4 nmol s-1/µg of protein, and the Kcat value was 0.92 ± 0.35 s-1 (Table I).


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  		TABLE I
The calculations were carried out using Sigma Plot (v8) with a kinetic module

The NTPase and RTPase assays were performed in a 96-well format as described under "Materials and Methods." The values for NTPase were calculated from 10 independent assays with different batches of enzymes, whereas the RTPase values represent the average of duplicate experiments with the same batch of enzyme.

 
Poly(A) Stimulation of the NTPase Activity of NS3FL—Our previous study indicated that the NTPase activity of the NS3{Delta}160 deletion mutant was stimulated by poly(A), which required not only the region between the 161-180 amino acid residues but also the 184RKRK motif (16). This conclusion was reached based on our results that the poly(A)-stimulated NTPase activity was abolished with the NS3{Delta}160(4M) mutant protein containing the 184RKRK -> 184QNGN mutation or with the deletion of an additional 20 amino acid residues from NS3{Delta}160 protein (NS3{Delta}180 protein), although the basal NTPase activity of either protein was unaffected. We sought to examine whether the basal NTPase activity of the NS3FL protein is also stimulated by poly(A) and whether the 184RKRK -> 184QNGN mutation has any effect on the poly(A)-stimulated NTPase activity. We expressed and purified the NS3FL(4M) mutant protein under conditions similar to those for NS3FL (data not shown) and assayed its NTPase activity. Fig. 4 shows that poly(A) stimulated the basal activity of NS3FL protein but not the basal activity of the NS3FL(4M) mutant. However, the degree of stimulation of the NTPase activity of NS3FL was only about 2-fold compared with 5-7-fold for the NS3{Delta}160. This can be explained due to a higher basal NTPase activity of NS3FL than the NS3{Delta}160. Moreover, the positively charged motif 184RKRK was also important for RNA-stimulated NTPase activity of NS3FL because mutation of this motif abolished this stimulation (Fig. 4). This result suggested that the 184RKRK motif is involved in RNA binding, and the resultant conformational change is required for stimulation of NTPase activity.



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  		FIG. 2.
Purification of NS3 proteins. The purification of NS3 proteins with an N-terminal His tag was as described previously for NS3FL (22) and as described under "Materials and Methods." A and B, Coomassie Blue-stained SDS-PAGE gels of purified proteins. A, lane M, protein molecular weight markers; lane 1, NS2BH(QR)-NS3FL; lane 2, NS2BH(QR)-NS3FL(H51A). B, lane 1, molecular size markers; lane 2, purified NS3FL (wild type). C, lane 1, molecular size markers; lane 2, purified NS5. D, lane M, molecular size markers; lane 1, NS2BH(QR)NS3FL after dialysis at pH 9.0; lane 2, the same protein as in lane 1 before dialysis. E, Western blot of the proteins on the gel in D with rabbit polyclonal anti-NS2B antibody (14). F, Western blot of the proteins on the gel in D with rabbit polyclonal anti-NS3 antibody (22).

 



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  		FIG. 3.
Analysis of NTPase activity of NS3FL. The NTPase assays of NS3FL with different substrate (ATP) concentrations in triplicate were performed as described under "Materials and Methods." From the Lineweaver-Burk plots (inset), the kinetic parameters were calculated as shown in Table I.

 
Characterization of the RNA Helicase of NS3FL Protein—Next, we examined the RNA helicase activities associated with NS3FL and NS3FL(4M) (Fig. 5). The helicase assay utilized synthetic double-stranded RNA substrate having a 3'-single-stranded terminus, formed by annealing of a synthetic 32P-labeled 30-mer RNA to a 15-mer complementary RNA according to the method described previously (44). We used a 100-fold excess of the enzyme to substrate in all the experiments shown in Fig. 5, A-D. The results shown in Fig. 5 indicated that the presence of the protease domain in the NS3FL had an enhancing effect on the RNA helicase activity compared with that of the protease domain-deleted NS3 (NS3{Delta}160) protein described previously (16). The NS3{Delta}160 protein was required at 2.7 µM for RNA unwinding of less than 5% of the substrate RNA (16). Interestingly, the mutation of the 184RKRK motif significantly reduced the RNA helicase activity of NS3FL (Fig. 5D). This is the first report for the function of this positively charged motif in RNA helicase activity of any flaviviral NS3 protein.



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  		FIG. 4.
The basal NTPase activity of NS3FL but not NS3FL(4M) mutant was stimulated by poly(A). The standard ATPase assays were carried out as described under "Materials and Methods" with purified NS3FL or NS3FL(4M) protein in the absence or presence of increasing concentrations of poly(A). The assays were done in triplicate in 96-well microtiter plates, and the average values of Kcat/s were plotted. {09200104-01}, NS3FL; {09200104-02}, NS3FL(4M).

 
We also examined whether the presence of the protease cofactor, NS2BH, in a binary complex with NS3FL (NS2BH(QR)/NS3FL), which was formed after cleavage of the QR{downarrow} site, had any effect on the RNA unwinding activity catalyzed by NS3FL. Our results showed that the presence of NS2BH cofactor domain at the N terminus of NS3FL protease domain in the binary complex did not have a significant enhancing effect on the RNA helicase activity of NS3FL. We surmised that in NS2BH(QR)/NS3FL, the activated protease in the binary complex had an opposing effect on the helicase activity as seen from the time course of RNA unwinding by NS3FL versus NS2BH(QR)/NS3FL. Therefore, we constructed the NS2BH(QR)-NS3FL (51His -> Ala) plasmid in which the protease domain was inactivated by mutagenesis of the catalytic triad residue, 51His -> Ala and purified the mutant protein as an uncleaved precursor (Fig. 2A). This protein had reproducibly, an enhanced RNA helicase activity over the NS3FL (Fig. 5, A versus B; see also E).



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  		FIG. 5.
RNA helicase assays of the various NS3FL proteins. The RNA helicase assays were performed using different NS3FL proteins as indicated (A-D). Samples were removed, and the reactions were stopped at various time points and analyzed by polyacrylamide gel electrophoresis as described under "Materials and Methods." The slower migrating and faster migrating bands represent double- and single-stranded RNA, respectively. In D, lane 3, the enzyme NS2BHQR/NS3FL was used as a positive control. The percent of double-stranded RNA substrate unwound was estimated by PhosphorImager (Molecular Dynamics) and plotted in each case (E). {blacktriangledown}, NS2BH-NS3FL(H51A); {circ}, NS3FL; , NS2BHQR/NS3; {triangledown}, NS3FL(4M). These experiments were repeated four times with each of the proteins. F, the helicase assays of the NS2BHQR/NS3FL protein were performed at two different substrate:enzyme ratios, 1:40 ({circ}) and 1:400 (). The data were plotted using the Sigma plot, version 8.

 
We sought to determine whether changing the ratio of the enzyme to substrate had any effect on the rate of unwinding. The unwinding of the substrate by the NS2BH(QR)/NS3FL protein was carried out at an [E:S] ratio of 40 and 400. As shown in Fig. 5F, increasing the enzyme to substrate ratio significantly increased the activity of the NS3 RNA helicase. These results are consistent with a recent study which revealed that a large excess of the HCV NS3 helicase was required for optimal unwinding of the substrate (35).

The NTPase Activity of NS3FL Is Stimulated by Interaction with NS5, the RNA-dependent RNA Polymerase—In DEN2-infected cells, NS3 and NS5 exist as a complex and are thought to be important components of the viral replicase involved in RNA replication (39). However, the functional consequence of this interaction and which step this complex is required for viral replication have not yet been established. In this study we asked whether the NS5 protein has any influence on the NTPase activity of NS3. The NTPase activity of NS3FL was assayed in the absence and presence of increasing amounts of NS5. The results shown in Fig. 6 indicate that NS5 stimulated the NTPase activity of NS3FL in a dose-dependent manner until the molar ratio of 1:1 was reached. At this point the addition of further amounts of NS5 had no effect on the NTPase activity of NS3. From these results we conclude that a 1:1 stoichiometric complex of NS3·NS5 complex is the functional NTPase. Next, we examined whether the basal NTPase activity of NS3FL(4M) mutant can also be stimulated by the addition of NS5. The results shown in Fig. 6 indicate that the NTPase activity of the NS3FL(4M) mutant lost the ability to be stimulated by NS5, suggesting that stimulation of basal NTPase activity by RNA and NS5 is intimately linked.

The 5'-RTPase Activity of NS3 Is Also Stimulated by Interaction with NS5—We previously reported that the NS3FL has 5'-RTPase, the cleavage of the {gamma}-{beta} phosphodiester bond of a triphosphorylated RNA (22), and the same active site was involved in NTPase and 5'-RTPase activities of NS3 (20, 22). The 5'-RTPase is the first enzymatic step required in the 5'-cap addition of viral RNA. The N-terminal region of NS5 has been reported to possess the 2'-O-methyltransferase, an activity involved in the formation of type I cap, and the crystal structure of this domain was reported previously (51). We surmised that the 5'-cap addition is carried out by an enzyme complex of NS3 and NS5 and that this interaction, which enhanced the NTPase activity of NS3, might also influence its 5'-RTPase activity. Fig. 7 shows that the addition of submolar amount of NS5 (50 nM) to 280 nM NS3 stimulated the RTPase activity of NS3 ~5-fold on a triphosphorylated RNA substrate (100 nucleotides in length). The apparent Km of DEN2 NS3 for triphosphorylated RNA substrate was 29.3 µM, and the Vmax was 0.65 nmol of substrate hydrolyzed/s. The apparent Kcat for the RTPase was 0.09/s, and the catalytic efficiency, Kcat/Km, was 3185 M-1 s-1. The turnover number of the RTPase activity was 10-fold lower than that of the NTPase activity of NS3FL, although the catalytic efficiency for the two reactions was approximately similar (Table I).



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  		FIG. 6.
The NTPase activity of wild type NS3FL but not NS3FL(4M) mutant is stimulated by the addition of NS5. The standard ATPase assays were carried out in the presence of purified NS3FL or NS3FL(4M) (140 nM) as described under "Materials and Methods" in the absence or presence of increasing concentrations of purified NS5. The assays were done in triplicate in 96-well plates, and the average values of Kcat/s were plotted. {09200104-01}, NS3FL; {09200104-02}, NS3FL(4M).

 
184RKRK Motif Is a Key Determinant for RNA Binding—Our results indicated that the 184RKRK motif is an important determinant for both RNA-stimulated NTPase (Fig. 4) and NS5-mediated stimulation of the NTPase (Fig. 6) and RTPase (data not shown) activities of NS3. These results suggested that this motif of NS3FL might be involved in binding to RNA and modulating these activities. To test this notion, we performed electrophoretic mobility shift assays using a 32P-labeled RNA fragment produced by T7 RNA polymerase-catalyzed in vitro transcription representing the sequence from the 5'-end of DEN2 genome (100 nucleotides in length) and purified NS3FL and NS3FL(4M) proteins. The results shown in Fig. 8 indicate that the wild type NS3FL bound to the RNA probe and retarded the mobility of NS3 protein (lane 2). However, the mutant NS3FL(4M) protein failed to bind to the probe (lane 3), which supported the notion that this motif is required for RNA binding and to modulate the RNA-stimulated NTPase and 5'-RTPase activities.



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  		FIG. 7.
The RTPase activity of NS3FL is stimulated by the addition of NS5. The RTPase activity assays of NS3FL were performed in duplicate in 96-well microtiter plates using a triphosphorylated RNA (100 nt) as the substrate as described under "Materials and Methods" in the absence or presence of purified NS5. The concentration of NS3FL was 280 nM and of NS5 was 50 nM, and the RNA substrate was at 5.6 µM. The average values of Kcat/s were plotted.

 



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  		FIG. 8.
Electrophoretic mobility shift assay for RNA binding activity of NS3FL or NS3FL(4M) mutant. The electrophoretic mobility shift assay was performed as described under "Materials and Methods" using a 32P-labeled RNA probe (100 nt). The binding reactions were analyzed by native polyacrylamide/agarose composite gel electrophoresis, and the RNA-protein complexes were visualized by autoradiography. Lane 1, free probe; lane 2, probe RNA + NS3FL; lane 3, probe RNA + NS3FL(4M). The arrow closer to the origin of the gel indicates the RNA probe-NS3FL complex.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we launched a detailed biochemical and kinetic analysis of the RNA-stimulated NTPase/RNA helicase and 5'-RNA triphosphatase activities of full-length DEN2 NS3 protein. We established the following key findings. 1) Full-length NS3 with the serine protease domain at the N terminus and the conserved motifs found in several RNA-stimulated NTPases/RNA helicases at the C-terminal region of the protease domain is a more active RNA helicase than the N-terminal-truncated or the glutathione S-transferase-NS3 fusion protein that was reported previously (20, 22, 52). 2) Moreover, the interaction of NS3FL with NS5 stimulated the NTPase in a dose-dependent manner up to the point when a 1:1 stoichiometry was reached, and beyond this point NS5 had no effect on the NTPase activity, suggesting that the NS3·NS5 complex is the functional unit for the NTPase activity of NS3, which is coupled to RNA unwinding (3). The positively charged motif 184RKRK plays an important role in RNA binding, and this motif modulates both the RNA-stimulated NTPase, RNA helicase, and the 5'-RTPase activities, all requiring interaction with RNA. Recently, the structure of RNA helicase of yellow fever virus has been solved, which suggests that the positively charged side of the helicase structure might play a role in single-stranded nucleic acid binding.2 However, the RKRK motif is not proximal to the NTP binding site nor to the presumed double-stranded RNA binding region.2 Because our data clearly show that the poly(A)-stimulated NTPase activity of NS3{Delta}160 (16) as well as NS3FL (this study) is abolished by mutation of RKRK -> QNGN, it is possible that the poly(A) binds to the interdomain cleft2 where single-stranded nucleic acid is bound as in other helicases including the HCV enzyme (53-55), and the resultant conformational change is involved in NTPase stimulation. The notion that a conformational change is required for NTPase stimulation is supported by our previous observation (16) that NS3{Delta}180 protein, which had a deletion of the N-terminal 180 amino acid residues, did not exhibit RNA-stimulated NTPase activity even though this protein has the positively charged motif situated close to the N terminus. Recent biochemical and kinetic analysis of HCV NS3 RNA helicase supports that multiple molecules of NS3 bound to the single-strand region or the junction of single-stranded and double-stranded regions of the substrate which seemed to be required for optimal unwinding (35). Blunt-end duplex is not an optimal substrate for unwinding by HCV NS3 helicase (44). Therefore, we propose that the positively charged motif in Flavivirus NS3 might play a role in initial binding of the enzyme to the single-stranded region of the substrate RNA before unwinding of the duplex region. Our observation that mutation of this positively charged motif significantly reduced the RNA helicase activity is also consistent with this notion.



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  		FIG. 9.
Sequence alignments of conserved, positively charged motifs in flaviviral NS3. The amino acid sequences of NS3 protein of various flaviviruses were deduced from the nucleotide sequences downloaded from GenBankTM. The nucleotide sequence of DEN2-NGC strain is from Irie et al. (6). The positively charged motifs are shown in bold. The accession numbers for the polyprotein sequences coded by the various Flavivirus genomes from which these conserved sequences were compiled are: DEN1, AB178040 [GenBank] ; DEN2, M29095 [GenBank] ; DEN2 strain 16681, U87411 [GenBank] ; DEN3 strain BR74886/02, AY679147 [GenBank] ; DEN4, M14931 [GenBank] ; Kunjin virus (KUN), D00246 [GenBank] ; Japanese encephalitis virus (JEV), NC_001437 [GenBank] ; West Nile virus (WNV), NC_001563 [GenBank] ; Murray valley encephalitis virus (MVEV) strain 1-51, AF161266 [GenBank] ; yellow fever (YF) virus strain 17D, X03700 [GenBank] ; YF strain Asibi, AY640589 [GenBank] ; YF strain Ivory Coast, AY603338 [GenBank] .

 
We examined the conservation of this positively charged motif in NS3 protein of other flaviviruses. As shown in Fig. 9, this motif is conserved across the flaviviral NS3 NTPase/RNA helicases, suggesting an important role in viral life cycle. Interestingly, the amino acid sequences in the C-terminal side of this positively charged motif are also conserved, suggesting that for a conformational change induced by single-stranded nucleic acid binding, additional amino acid residues might also be involved. However, in contrast to our results, in another study using glutathione S-transferase fusion proteins of DEN2 NS3 and mutants within and outside the helicase motifs, the authors concluded that mutation of this positively charged motif enhanced the RNA helicase activity (52). The reason for this discrepancy is unclear and might be attributed to differences in the nature of the recombinant proteins and RNA substrates used in the two studies. Our observation that mutation of this positively charged motif abolished the NS5-mediated stimulation of NTPase and 5'-RTPase activities of NS3 when the two proteins are present in stoichiometric amounts suggested that this motif is also involved in stabilization of NS3/NS5 interaction to form a functional complex. Further work is needed to establish the role of this motif and the conserved amino acid residues juxtaposed with this motif in NS3/NS5 interaction in the presence and absence of RNA.

Note Added in Proof—Recently, Zhang et al. communicated to us that, in HCV NS3, the protease domain enhanced the activitiy of the helicase domain, which was further stimulated by interaction with HCV RNA-dependent RNA polymerase (Zhang, C., Zhaohui, C., Kim, Y-C., Kumar, R., Yuan, F., Shi, P-Y., Kao, C., and Luo, G. J. (2005) J. Viral. 79, in press).


   FOOTNOTES
 
* This work was supported from NIAID, National Institutes of Health Grants AI32078, AI54776 and AI45623. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Georgetown University School of Medicine, 3900 Reservoir Rd., Washington, D. C. 20057. Tel.: 202-687-2092; Fax: 202-687-1800; E-mail: rp55{at}georgetown.edu.

1 The abbreviations used are: DEN2, dengue virus type 2; FL, full-length; HCV, hepatitis C virus; NTPase, nucleoside triphosphatase; NS, non-structural (proteins that are expressed in infected cells but are not part of virions); 5'-RTPase, 5'-RNA triphosphatase; nt, nucleotide(s). Back

2 J. Smith, Purdue University, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kevin D. Raney and Dr. Craig Cameron for the gift of HCV NS3 RNA helicase during the initial stage of this study. We thank Dr. Janet Smith for communication on the structural aspects of YF RNA helicase before publication.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Campbell, G. L., Marfin, A. A., Lanciotti, R. S., and Gubler, D. J. (2002) Lancet Infect. Dis. 2, 519-529[CrossRef][Medline] [Order article via Infotrieve]
  2. Roehrig, J. T., Layton, M., Smith, P., Campbell, G. L., Nasci, R., and Lanciotti, R. S. (2002) Curr. Top. Microbiol. Immunol. 267, 223-240[Medline] [Order article via Infotrieve]
  3. Brinton, M. A. (2002) Annu. Rev. Microbiol. 56, 371-402[CrossRef][Medline] [Order article via Infotrieve]
  4. Gubler, D. J. (1998) Clin. Microbiol. Rev. 11, 480-496[Abstract/Free Full Text]
  5. Halstead, S. B. (2002) Curr. Opin. Infect. Dis. 15, 471-476[Medline] [Order article via Infotrieve]
  6. Irie, K., Mohan, P. M., Sasaguri, Y., Putnak, R., and Padmanabhan, R. (1989) Gene (Amst.) 75, 197-211[CrossRef][Medline] [Order article via Infotrieve]
  7. Lindenbach, B. D., and Rice, C. M. (2003) Adv. Virus Res. 59, 23-61[CrossRef][Medline] [Order article via Infotrieve]
  8. Chambers, T. J., Grakoui, A., and Rice, C. M. (1991) J. Virol. 65, 6042-6050[Abstract/Free Full Text]
  9. Falgout, B., Pethel, M., Zhang, Y. M., and Lai, C. J. (1991) J. Virol. 65, 2467-2475[Abstract/Free Full Text]
  10. Wengler, G., Czaya, G., Farber, P. M., and Hegemann, J. H. (1991) J. Gen. Virol. 72, 851-858[Abstract/Free Full Text]
  11. Zhang, L., Mohan, P. M., and Padmanabhan, R. (1992) J. Virol. 66, 7549-7554[Abstract/Free Full Text]
  12. Arias, C. F., Preugschat, F., and Strauss, J. H. (1993) Virology 193, 888-899[CrossRef][Medline] [Order article via Infotrieve]
  13. Falgout, B., Miller, R. H., and Lai, C.-J. (1993) J. Virol. 67, 2034-2042[Abstract/Free Full Text]
  14. Clum, S., Ebner, K. E., and Padmanabhan, R. (1997) J. Biol. Chem. 272, 30715-30723[Abstract/Free Full Text]
  15. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P., and Blinov, V. M. (1989) Nucleic Acids Res. 17, 4713-4730[Abstract/Free Full Text]
  16. Li, H., Clum, S., You, S., Ebner, K. E., and Padmanabhan, R. (1999) J. Virol. 73, 3108-3116[Abstract/Free Full Text]
  17. Suzich, J. A., Tamura, J. K., Palmer Hill, F., Warrener, P., Grakoui, A., Rice, C. M., Feinstone, S. M., and Collett, M. S. (1993) J. Virol. 67, 6152-6158[Abstract/Free Full Text]
  18. Warrener, P., Tamura, J. K., and Collett, M. S. (1993) J. Virol. 67, 989-996[Abstract/Free Full Text]
  19. Wengler, G., and Wengler, G. (1991) Virology 184, 707-715[CrossRef][Medline] [Order article via Infotrieve]
  20. Benarroch, D., Selisko, B., Locatelli, G. A., Maga, G., Romette, J. L., and Canard, B. (2004) Virology 328, 208-218[CrossRef][Medline] [Order article via Infotrieve]
  21. Wengler, G., and Wengler, G. (1993) Virology 197, 265-273[CrossRef][Medline] [Order article via Infotrieve]
  22. Bartelma, G., and Padmanabhan, R. (2002) Virology 299, 122-132[CrossRef][Medline] [Order article via Infotrieve]
  23. Shuman, S. (2002) Nat. Rev. Mol. Cell Biol. 3, 619-625[CrossRef][Medline] [Order article via Infotrieve]
  24. Rocak, S., and Linder, P. (2004) Nat. Rev. Mol. Cell Biol. 5, 232-241[CrossRef][Medline] [Order article via Infotrieve]
  25. Silverman, E., Edwalds-Gilbert, G., and Lin, R. J. (2003) Gene (Amst.) 312, 1-16[CrossRef][Medline] [Order article via Infotrieve]
  26. Gwack, Y., Kim, D. W., Han, J. H., and Choe, J. (1996) Biochem. Biophys. Res. Commun. 225, 654-659[CrossRef][Medline] [Order article via Infotrieve]
  27. Kim, D. W., Gwack, Y., Han, J. H., and Choe, J. (1995) Biochem. Biophys. Res. Commun. 215, 160-166[CrossRef][Medline] [Order article via Infotrieve]
  28. Jin, L., and Peterson, D. L. (1995) Arch. Biochem. Biophys. 323, 47-53[CrossRef][Medline] [Order article via Infotrieve]
  29. Tai, C. L., Chi, W. K., Chen, D. S., and Hwang, L. H. (1996) J. Virol. 70, 8477-8484[Abstract]
  30. Kim, D. W., Kim, J., Gwack, Y., Han, J. H., and Choe, J. (1997) J. Virol. 71, 9400-9409[Abstract]
  31. Levin, M. K., and Patel, S. S. (1999) J. Biol. Chem. 274, 31839-31846[Abstract/Free Full Text]
  32. Pang, P. S., Jankowsky, E., Planet, P. J., and Pyle, A. M. (2002) EMBO J. 21, 1168-1176[CrossRef][Medline] [Order article via Infotrieve]
  33. Levin, M. K., Gurjar, M. M., and Patel, S. S. (2003) J. Biol. Chem. 278, 23311-23316[Abstract/Free Full Text]
  34. Levin, M. K., Wang, Y. H., and Patel, S. S. (2004) J. Biol. Chem. 279, 26005-26012[Abstract/Free Full Text]
  35. Tackett, A. J., Chen, Y., Cameron, C. E., and Raney, K. D. (2005) J. Biol. Chem. 280, 10797-10806[Abstract/Free Full Text]
  36. Warrener, P., and Collett, M. S. (1995) J. Virol. 69, 1720-1726[Abstract]
  37. Gu, B., Liu, C., Lin-Goerke, J., Maley, D. R., Gutshall, L. L., Feltenberger, C. A., and Del Vecchio, A. M. (2000) J. Virol. 74, 1794-1800[Abstract/Free Full Text]
  38. Utama, A., Shimizu, H., Morikawa, S., Hasebe, F., Morita, K., Igarashi, A., Hatsu, M., Takamizawa, K., and Miyamura, T. (2000) FEBS Lett. 465, 74-78[CrossRef][Medline] [Order article via Infotrieve]
  39. Kapoor, M., Zhang, L., Ramachandra, M., Kusukawa, J., Ebner, K. E., and Padmanabhan, R. (1995) J. Biol. Chem. 270, 19100-19106[Abstract/Free Full Text]
  40. Westaway, E. G., Mackenzie, J. M., and Khromykh, A. A. (2003) Adv. Virus Res. 59, 99-140[CrossRef][Medline] [Order article via Infotrieve]
  41. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
  42. Zhao, L. J., Zhang, Q. X., and Padmanabhan, R. (1993) Methods Enzymol. 217, 218-227[Medline] [Order article via Infotrieve]
  43. You, S., and Padmanabhan, R. (1999) J. Biol. Chem. 274, 33714-33722[Abstract/Free Full Text]
  44. Tackett, A. J., Wei, L., Cameron, C. E., and Raney, K. D. (2001) Nucleic Acids Res. 29, 565-572[Abstract/Free Full Text]
  45. Lawrence, R. (1974) Ann. Clin. Biochem. 11, 234-237[Medline] [Order article via Infotrieve]
  46. Bartels, P. C., and Roijers, A. F. (1975) Clin. Chim. Acta 61, 135-144[Medline] [Order article via Infotrieve]
  47. Lam, A. M., Keeney, D., Eckert, P. Q., and Frick, D. N. (2003) J. Virol. 77, 3950-3961[Abstract/Free Full Text]
  48. Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K., Chan, Y. C., and Fu, J. (1998) Virology 246, 409-417[CrossRef][Medline] [Order article via Infotrieve]
  49. Ackermann, M., and Padmanabhan, R. (2001) J. Biol. Chem. 276, 39926-39937[Abstract/Free Full Text]
  50. Melese, T., Xue, Z. X., Stempel, K. E., and Boyer, P. D. (1988) J. Biol. Chem. 263, 5833-5840[Abstract/Free Full Text]
  51. Egloff, M. P., Benarroch, D., Selisko, B., Romette, J. L., and Canard, B. (2002) EMBO J. 21, 2757-2768[CrossRef][Medline] [Order article via Infotrieve]
  52. Matusan, A. E., Pryor, M. J., Davidson, A. D., and Wright, P. J. (2001) J. Virol. 75, 9633-9643[Abstract/Free Full Text]
  53. Kim, J. L., Morgenstern, K. A., Griffith, J. P., Dwyer, M. D., Thomson, J. A., Murcko, M. A., Lin, C., and Caron, P. R. (1998) Structure 6, 89-100[Medline] [Order article via Infotrieve]
  54. Kim, J. W., Seo, M. Y., Shelat, A., Kim, C. S., Kwon, T. W., Lu, H. H., Moustakas, D. T., Sun, J., and Han, J. H. (2003) J. Virol. 77, 571-582[Medline] [Order article via Infotrieve]
  55. Yao, N., Reichert, P., Taremi, S. S., Prosise, W. W., and Weber, P. C. (1999) Structure Fold. Des. 7, 1353-1363[Medline] [Order article via Infotrieve]

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