Multiple enzyme activities of flavivirus proteins

R. Padmanabhan*, N. Mueller*, E. Reichert*, C. Yon*, T. Teramoto*, Y. Kono*, R. Takhampunya*^, S. Ubol^, N. Pattabiramanf, B. Falgout$, V. K. Ganesh§ and K. Murthy§

* Department of Microbiology and Immunology and f Lombardi Cancer Center, Georgetown University School of Medicine, Washington DC, USA, $ CBER, FDA, Bethesda, MD, USA, § University of Alabama-Birmingham, AL, USA and ^ Department of Microbiology, Mahidol University, Bangkok, Thailand

Abstract. Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified ~200 compounds having >50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito-or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

2006 New treatment strategies for dengue and other flaviviral diseases. Wiley, Chichester (Novartis Foundation Symposium 277) p 74—86

Studies from several laboratories have strongly implicated a complex interplay of viral non-structural proteins as well as cellular proteins, in modulating the process of viral replication (Lindenbach & Rice 2003). Since the title of my presentation is with regard to multiple enzyme activities of flaviviral non-structural proteins, I give below a summary of the work already published from our laboratory as well those reported by others as a prelude to the more recent ongoing research. Our efforts have mainly focused on functional analysis of two viral proteins, NS3 and

Protease RKRK NTPase/Helicase

GAGKT PTRDEAH TAT SEMG QRRGRIGR

FIG. 1. Multiple domains of NS3. Protease domain is at the N-terminus within 170 amino acid residues. H, D and S are at position 51, 75 and 135. Six conserved motifs comprising the RNA helicase domain are shown as open vertical boxes. Black box is a stretch of basic amino acids.

NS5. NS3 is a paradigm of multifunctional proteins (Fig. 1). The N-terminal (180 amino acid) region is known to have a serine protease domain that requires the NS2B cofactor for protease activity (Chambers et al 1991, Falgout et al 1991, Wengler et al 1991, Zhang et al 1992). The two components of the protease interact and form a stable complex in flavivirus-infected cells (Arias et al 1993, Chambers et al 1993). The NS2B is an endoplasmic resident integral membrane protein and the hydropathy plot of the protein shows a conserved hydrophilic domain of NS2B (NS2BH) flanked by hydrophobic regions (Clum et al 1997). The deletion analysis of NS2B indicated that a conserved hydrophilic domain of -40 residues (Fig. 2A) is sufficient for protease activity in vivo (Falgout et al 1993) as well as in vitro (Clum et al 1997). When the His-tagged NS2BH domain linked to serine protease domain is expressed in Escherichia coli, it undergoes a cis cleavage at the junction of the NS2BH-NS3-pro precursor and the protease can be purified as a complex by metal affinity column. The protease is active in cleaving a radiolabelled NS4B-NS5 precursor substrate or chromogenic and fluorogenic peptide substrates (Leung et al 2001, Li et al 2005, Yusof et al 2000). The crystal structure of the dengue virus (DENV)-2 NS3 protease domain alone in the absence of the NS2BH cofactor domain has been determined (Murthy et al 1999). The structure of the protease domain reveals that the N- and C-terminal P barrels are six-stranded and the catalytic triad consists of His51, Asp75, and Ser135. Molecular modelling of a tetrapep-tide into the substrate binding cleft indicates that the protease domain has a shallow substrate binding pocket with no extensive side chain interactions beyond P2 and P2' residue, suggesting that interaction with the cofactor NS2B results in a dramatic change in conformation that results in more extensive interactions with the substrate beyond P2 and P2'. In fact, the protease activity of NS3-pro is enhanced 104-fold against the tripeptide substrate by the interaction with the NS2BH whereas it has negligible effect on hydrolysis of a substrate with a single Arg such as W-benzoyl-L-Arg-p-nitroanilide (BAPNA). This structure of the protease domain could explain the effects of mutations of the protease domain previously reported (Valle & Falgout 1998). The crystal structure of the NS3 protease

Protease RKRK NTPase/Helicase

A DEN4 LEKAANVQWDEMADITG3S DEN3 VEKAADVTWEE EAEQTGVS DEN2 LERAADVKWEDQAEI3G3S DEN1 LE KAAEV S W E E E AE H 3 G A S YFV LKKLGEVSWEEEAEI3G3S MVE LERAADVSWEAGAAITGTS SLE IEKAADITWEQNAEITGTS JEV LERAADISWEMDAAITGSE WNV IERTADITWE5 DAEITG3 S KUN IERTADISWEG DAE ITGS £ TBE AEWSCCVEWYPELVNEGGE Left half

PIIEVKQDEDGSESIRDVEET H NLMIT VDDDGT>I RIK DDE IE PI LSI"13 E3G3M31KKEEEE HNILVEVQDDGTMKIKDEERD ARYDVAL3 EQGE FKLLSSEKV ERLDVQLDDDGDFHLLNDPGV PRLDVDLD3 HGNFKLLNDPGA RRLDVKLDD DGDFHLIDDFGV ERVDVRLDDJGNFQLMNDPGA ERVDVRLDD DGNFQLMNDPGA VSLRVRQ3AMGNFHLT EL E KE Right half

Den NSSpro JEV NS3pro SUV NS3pco YFV NS3pro Consensus

Den NS3pco JEV NS3pro WNV NS3pro YFV HS3pro Consensus

Den NS3pro (99) JEV NSSpro (99) WNV NS3pro (99) YFV NS3pro(101) Consensus (101)

Den NSSpiro (148) JEV NS3pro(148) WNV NS3pro(148) YFV NS3pro(151) Consensus(151)

AGVLWDVFSPPFVGKAHj- LEDGAYRIKQKGILGYSOIC^JVYKEGTFil™ - GGVTWD TF SP K PC S KGD TTTGVY RIMASG1L.GTYQA.GV SVMYENVF1 • -GGVLWDTFSPtSYKKGD-bTTGVYslMTRGU GSY0AGAGVMVEGVF1IT SGDVLWD IF SPKIISECEHLEDGIlfSI FQSTFLGASQE 3V3VAQGGVF1 i T GGVLWDTPSPKPI KGD TTTGVYRIMQRGILGSYQAGVGVM EGVFHT 50 60 70 80 90 98

K ■: rVTRGAVlMI iKGKRIE ? SKA3VKSDL I SI" - JGWKLEGEWKEGEEY: VL L' : TTRGAAIMSSESFLTPY'-iS SVK2CR IA.V JIG PWR FDRKWNG TDD VQV I L" ;T" K ;.;iALMSGEGRLD?Y;iG£Vi;2DRLCi ¿SPKKLQHKKNGKDE'. -MI Iff-::; VTOGAFLVRCGKKLIPSWASVieDLVAY J®SWKLEGRjfDGEEE Vy LI LWHVTRGAALMSGGKKLD PYWASVKEDRIAYGGPWKLEGKWNG DEVQVI

ALEPGKNPRAVlQTKPGliFST-NTGTIGAVSLDPSPGTSSSPIVDKKGfiVV WEPGKAAVN3 . : iTGVFRT-PFGEVi : .VSLDYPRG: S3SFTLDSNGDII jgVEPGKNVKNVQTEF GVFXT-PEGE !(.-Tl DYPTG: rGSPIVDKNGDVI AAVPGKNWNVGTE F SLFKYKNGGE IGAVALDrPSG 1S G 5 PIVNHNGEV: WEPGKNWiiVCTKPGLFKT P GEIGAVSLEYPSGTSGSPIVDKNGDVI

150 160 170 180

GLYGNGWTRSCftWSAIAQTSKSISaNPE IED3IF P.KRRL GLY GNGVELGDGSYVSAIVQGljRQEE = VP EAYT PNMLRIIQI GLYGNGVHi?NGSYISAIVQG3RME*PA?AGFE?EMLRKKQI j LY G1JG ILVGDNSEVSA ISQTSVKE E GKE E LQ£ IPTMLKKG-GLYGNGVrLGDGSYVSAIVC'TER EEP PEIFEP MLRKKQI

FIG. 2. Sequence alignments of the two-component protease, NS2BH-NS3-pro.

domain in a 2 : 1 complex with a double-headed mung bean Bowman-Birk inhibitor (MbBBI) is also known (Murthy et al 2000). The MbBBIs are hydrolysed by serine proteases in a standard manner; however, the reaction is completely reversible with no appreciable product release. Both NS3-pro and NS2BH/NS3-pro complex are inhibited by MbBBI using the BAPNA as the substrate with a IC50 value of -1.8 n.M. The structure reveals that the P1 Arg is bound to a bifurcated S1 pocket in a distinct manner (Murthy et al 2000). Although the overall structural features of the NS3-pro/MbBBI complex are in agreement with many other serine protease/inhibitor complexes, there are some differences with respect to interactions made by P1 Arg and Lys with the residues in the S1 pocket of NS3-pro, including the presence of the Arg at P1 in two different conformations and also large conformational changes of some of the residues of NS3-pro that interact with the Arg. However, since the structure of the NS3-pro and MbBBI was obtained in the absence of the NS2BH cofactor the interpretation of these results is made with caution. Based on the bifurcated recognition mode of the P1 Arg by the S1 pocket of the NS3-pro, we reasoned that compounds with terminal biguanidino groups, which could be superimposed on the guanidino groups of the bifurcated Arg side chain, might be possible candidates as selective inhibitors of DENV-2 NS2B :NS3-pro. In fact, of the three compounds tested, only one compound inhibited with a reasonable potency (35 and 44 |iM for West Nile, WNV, and DENV proteases, respectively). Two other compounds with a single guanidino group inhibited DENV-2 and WNV proteases with Ki values in the range of 13-23 |iM but also inhibited trypsin with IK values in the 3.2 to 4 |iM range (Ganesh et al 2005).

The spacer region between the NS2BH and the NS-pro is not critical for protease activity. An active protease was expressed in E. coli in a non-cleavable form by substitution of the natural spacer with a G4-S-G4 linker (Leung et al 2001). Some substrate analogues were found to inhibit the DENV protease in a competitive manner (Leung et al 2001). A comparative study of the substrate specificity of all four DENV NS3-pro was reported using tetra peptide libraries (Li et al 2005). Their results indicated that a strong preference for Arg/Lys at P1 whereas for P2-P4 sites, the order of preference was P2: Arg>Thr>Gln/Asn/Lys, P3: Lys>Arg>Asn>, P4: Nle>Leu>Lys>Xaa. At the prime sites, small and polar residues were preferred at P1' and P3', whereas the P2' and P4' sites had minimal effect. The N-terminal (P6-P1) cleavage site peptides were also found to inhibit the protease activity in a competitive manner with K; values in the range of 6712 |iM. The peptides from the P1'-P5' region had no inhibitory effect (Chanpra-paph et al 2005).

The region C-terminal to the protease domain of NS3 has conserved domains found in the DEXH family of NTPases/RNA helicases (Fig. 1). The motif GxGKS/T (domain I) and domain II are required for the ATPase activity of NS3. The NTPase activity of NS3, involved in the hydrolysis of the y-ipa phosphoric anhydride bond (shown by an arrow) of NTP has been reported for several flavi-viruses including DENV-2 (Li et al 1999, Cui et al 1998, Benarroch et al 2004, Yon et al 2005). Several viral NTPase activities are stimulated by the addition of single-stranded RNA. The presence of conserved RNA helicase motifs in flavivi-rus NS3 is consistent with its postulated role in viral replication in a key step involving unwinding of the double-stranded RNA replicative form. The RNA

helicase activity has been shown for DENV-2 and Japanese encephalitis viruses (Li et al 1999, Utama et al 2000, Benarroch et al 2004, Yon et al 2005). However, the role of the RNA-stimulated NTPase/RNA helicase activity of NS3 in viral life cycle has not been established for any flavivirus. In addition, NS3 has the 5'-RNA triphosphatase activity (5'-RTPase), capable of hydrolysing the y^Pa phosphoric anhydride bond (shown by an arrow) of triphosphorylated RNA as shown for full-length DENV-2 NS3 expressed and purified from E. coli (Li et al 1999, Benarroch et al 2004, Yon et al 2005). The 5'-RTPase is the first of the four sequential enzymatic reactions that are involved in the addition of 5'-cap to RNA.

The multifunctional NS3 protein exists in a complex with NS5 (Kapoor et al 1995), which itself has two enzyme activities, the 5'-RNA O-methyltransferase involved in 5'-capping and the RNA-dependent RNA polymerase required for viral RNA replication in flavivirus-infected cells (Fig. 3). After hydrolysis of the y-phosphate moiety of triphosphosphorylated RNA (the intrinsic activity of NS3) two additional enzyme activities are required for formation of the 5'-cap, the guanylyltransferase and the two 5'-RNA methyltransferase activities, respectively. Since the flavivirus genomes have type I cap structure at the 5'-end, two steps are involved in the 5'-cap addition: methyl transfer to 7-methylG and to 2'-OH of the first 5'-terminal nucleotide of RNA. The N-terminal domain of NS5 was shown to catalyse the transfer of methyl group from S-adenosylmethionine to 2'-OH and the crystal structure of this domain was reported (Egloff et al 2002). Viruses that replicate in the cytoplasm, in general, provide their own capping machinery and the viral proteins that are involved have multiple functions. Mutational analysis of DENV-2 NS3 indicates that the active site of the NTPase and the 5'-RTPase share one enzymatic function i.e. removal of y-phosphate moiety of either ATP (NTPase) or the RNA substrate (5'-RTPase) (Bartelma & Padmanabhan 2002, Benarroch et al 2004).

The C-terminal domain of NS5 has the RNA-dependent RNA polymerase (RdRP) activity which is required for RNA synthesis in vitro as shown using cellfree systems that utilize endogenous (Grun & Brinton 1986, Chu & Westaway 1987, Uchil & Satchidanandam 2003b) or exogenous viral RNA templates (You & Padmanabhan 1999, You et al 2001, Nomaguchi et al 2003b). According to a current model for viral replication, the synthesis of progeny RNA(+) strands

MTase motifs

Conserved motifs in RdRP

FIG. 3. Flavivirus NS5 is a RNA-dependent RNA polymerase and 2'-O-methyltransferase.

Conserved motifs in RdRP

FIG. 4. Replication model for flavivirus RNA. The simplistic model is based on studies on the intracellular forms of DENV-2 and KUNV-infected cells. Three forms were identified: replicative intermediates (RI), replicative form (RF) and virion RNA (vRNA). The viral replicase and the vRNA (+) template are in association with membranes. The synthesis of progeny vRNA occurs in a semiconservative and asymmetric manner on a recycling RF (middle structure) and RI (bottom) templates.

FIG. 4. Replication model for flavivirus RNA. The simplistic model is based on studies on the intracellular forms of DENV-2 and KUNV-infected cells. Three forms were identified: replicative intermediates (RI), replicative form (RF) and virion RNA (vRNA). The viral replicase and the vRNA (+) template are in association with membranes. The synthesis of progeny vRNA occurs in a semiconservative and asymmetric manner on a recycling RF (middle structure) and RI (bottom) templates.

occurs via asymmetric and semiconservative replication on a template of dsRNA as a replicative intermediate (RI) or replicative form (RF), which serve as recycling templates (Fig. 4). This in vitro assay is useful to detect the intracellular forms of DENV-2 replicative RNA when infected cells were treated with inhibitors of viral replication. The exogenous subgenomic RNA template-dependent and template-specific replication system established in our laboratory has been useful in defining the requirements for minus and plus strand RNA synthesis. The cell lysate system gave the first evidence for long range interaction involving the conserved self-complementary 5'- and 3'-cyclization (CYC) motifs (You & Padmanabhan 1999) as well as the 5'- and 3'-stem-loop structures that are required for (—) strand RNA synthesis. Physical interaction between the 5'- and 3'-ends was shown by psoralen-UV cross-linking (You et al 2001) and by atomic force microscopy (Alvarez et al 2005). Recombinant DENV-2 or WNV NS5 alone expressed and purified from E. coli can synthesize (—)RNA in vitro (Ackermann & Padmanabhan 2001, Nomaguchi et al 2003a, 2003b) which also requires a functional interaction between the 5'- and 3'-ends (You & Padmanabhan 1999, Khromykh et al 2001, Corver et al 2003, Lo et al 2003).

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