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mutational studies that have shown that mutating both Asp129 and Tyr150 to Ala is necessary to inactivate the protease.101 However, neither of these structures contains either NS2B or NS2B(H), making it dif cult to determine the mechanism of activation and the conformation of the fully activated protease. The strongly hydrophilic nature of NS2B(H) indicates that NS2B/NS3-pro interaction is likely to be dominated by hydrophilic contacts, some evidence for which has been obtained from mutagenesis studies on the YFV protease.102 Using the NS2B(H)/NS3-pro mimic of the dengue protease, and NS3-pro domain alone, it has been demonstrated103 that NS3-pro can hydrolyze the chromogenic substrate, Arg-p-nitrophenylanilide, ef ciently but was essentially inactive on uorogenic tripeptide substrate, Boc-Gly-Arg-Arg-AMC (7-amido-4-methylcoumarin). Furthermore, NS2B(H) activates NS3-pro by approximately four orders of magnitude in hydrolysis of tripeptide substrates, while it has little effect on hydrolysis of substrates that possess only a P1 side chain, suggesting that interactions between the P1 residue and the enzyme are not strongly dependent on NS2B. These kinetic results were used to suggest two plausible hypothetical mechanisms for activation of Den2 NS3pro by NS2B. The rst posits conformational changes induced in NS3-pro by interaction with NS2B, which induces greater structural complementarity of the binding pockets in the former for the side chains of the substrate. The second mechanism postulates a direct interaction of NS2B with substrate side chains, resulting in greater stabilization of the enzyme activator substrate ternary complex.103 Both mechanisms have parallels in functioning of other two-component proteases.87,98 Although a modeling study104 has proposed structural details for NS2B NS3-pro interaction, in the absence of supportive experimental data, the molecular mechanism of activation of NS3-pro by NS2B is currently obscure. Despite reservations about the eventual signi cance of the MbBBI complex in aviviral polyprotein processing, the two structures Den2 NS3-pro94 and its complex with MbBBI100 are the only two aviviral protease structures that are currently available for use in structure-assisted design of inhibitors. Proteases are ubiquitous in biological processes and have been considered to be attractive targets for therapy of a variety of viral and nonviral diseases.105 However, because all proteases of a given class share common mechanistic features and differ only in their choice of substrates, making strongly selective or speci c inhibitors for a given protease is a challenging task. One generally adopted strategy in a search for speci c inhibitors is to modify a speci c substrate, commonly a short peptide, through substituting the peptide bond by a nonhydrolyzable analogue. Several a-keto amide analogues of peptide substrate-derived sequences at polyprotein cleavage sites have been reported,106 tested against a covalently linked NS2B-3 construct of Den2. The hexapeptide analogue based on the NS3/4A site had the best Ki of 47 mM. The same study also reported an irreversible inhibition by an aldehyde analogue with a Ki of 16 mM.106 Although there are no structural data at present, these inhibitors are expected to bind and behave as nonhydrolyzable substrates. Appropriate three-dimensional structures of inhibited complexes generally provide useful data for design of inhibitors using methods of structure-assisted methods.107 Although it is tempting, in structural terms, to use the bifurcated Arg conformation
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STRUCTURAL BIOLOGY OF FLAVIVIRAL REPLICATION
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in the MbBBI complex100 in designing selective inhibitors, the uncertain physiological relevance of this structure suggests caution. There is no other structural platform that can currently be used in structure-assisted design of aviviral serine protease inhibitors. However, with the increasing interest in aviviral enzymes, a suitable template structure might soon become available. Among alternative strategies that have been suggested for inhibiting aviviral proteases is the possibility of disrupting the interaction between NS3-pro and NS2B.101 Such an approach has also been suggested in the case of HCV protease.98 This strategy may be more effective for aviviral proteases than for HCV because of the greater enhancement of the activity of the former by NS2B compared to that of HCV NS3 protease by NS4A. While the activity of HCV NS3 protease is enhanced by a factor of 3 100 by NS4A, depending on the cleavage site,108 Den2 NS3-pro is activated by 4 5 orders of magnitude by NS2B103 and the WNV enzyme by a similar magnitude (R. Padmanabhan, personal communication). Thus, the aviviral proteases might be more critically dependent on their activation factors for attaining physiologically meaningful levels of activity. Evidence obtained from measuring the change in activation levels of Den2 NS2B/NS3-pro, as a function of NaCl concentration,103 and the strongly hydrophilic nature of the sequence of NS2B(H)70,75,91 indicate that electrostatic forces might make a signi cant contribution to NS3-pro NS2B interaction in aviviruses. In addition, mutational studies on YFV102 and Den2 (B. Falgout, personal communication) have suggested that not all residues of NH2B(H) are equally important for interaction with NS3pro. While this and other information could potentially be used in exploring peptides that might antagonize the NS2B(H) NS3-pro interaction, no published data appears to be currently available. Clues to nonpeptide molecular entities that could disrupt this interaction must await a structure that incorporates NS2B or NS2B(H). The RNA helicase activity of NS3 is encoded in the carboxyl terminal two-thirds of the protein and this region has signi cant sequence similarity to the DEXd/H superfamily of RNA helicases.109,110 A prerequisite for helicase activity is an RNAstimulated ATPase activity that provides the energy for helix unwinding. NS3 proteins from JEV,111,112 YFV,113 WNV,83 and Den282 have been shown to express this activity. Mutational disruption of helicase activity has been used to demonstrate the essential nature of this enzyme in aviviral replication.114 The C-terminal regions of aviviral NS3 proteins also express an RNA triphosphatase (RTPase) activity, which might be important for 50 cap addition.80 Both the NTPase and the RTPase activities are sensitive to Mg2 , ionic strength, and nonhydrolyzable ATP analogues, as demonstrated for the WNV80 and Den2115 enzymes. In addition, work on Den2 NS3115 has established that mutation of Lys199, part of the presumed nucleotide binding site, abolishes both NTPase and RTPase activities. No threedimensional structure has been reported for this part of a aviviral protein, but structures of the closely related hepatitis C virus NS3 helicase, as well as its complexes with nucleic acids, are available.98,116,117 The structure of the entire NS3 protein of the hepatitis C virus protein has also been reported.118 Viral helicases, due to their mandatory involvement in one of the central events in viral
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