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Short Communication |
1 School of Molecular and Microbial Sciences, University of Queensland, Brisbane, QLD 4072, Australia
2 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia
Correspondence
Paul R. Young
p.young{at}uq.edu.au
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ABSTRACT |
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Supplementary material is available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Since the introduction of WNV to New York in 1999, the virus has spread rapidly throughout North America and into Canada, Mexico, Central America and the Caribbean (Gould & Fikrig, 2004; Komar & Clark, 2006), causing more than 900 fatalities and an estimated 280 000 infections in humans (http://www.cdc.gov/ncidod/dvbid/westnile/index.htm). Currently, there is no vaccine or antiviral therapy available for the prevention or treatment of human WNV infection (van der Meulen et al., 2005).
Flaviviruses are small, enveloped viruses with a single-stranded, positive-sense 11 kb RNA genome. While no effective antiviral therapy exists for any flavivirus, the viral protease within NS3 has attracted attention as a potential target for inhibitor development. This protease plays an essential role in the cleavage of the viral polyprotein precursor to create 10 functional proteins: three structural (C, prM and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Processing of the polyprotein is believed to occur in the viral induced convoluted membranes/paracrystalline arrays (CM/PC), which are continuous with the rough endoplasmic reticulum (Westaway et al., 1997). The viral protease recognizes and cleaves the C-terminal side of the highly conserved dibasic residue sequences located at the coding junctions, NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 and also at the C terminus of C and NS4A.
NS3 is a multifunctional protein, comprising two distinct functional domains. The protease comprises the N-terminal 184 aa residues of NS3, while the C terminus contains helicase, nucleoside triphosphatase and RNA triphosphatase activities (Chambers et al., 1990b; Gorbalenya et al., 1989; Wengler, 1991). The NS3 protease is only active as a heterodimeric complex with its cofactor, NS2B. For dengue type 2 (DEN2) protease, a 40 residue central hydrophilic domain, NS2B54–93, was found to be the minimum sequence required for protease activity and it was thought to interact directly with NS3 (Falgout et al., 1993). The remainder of NS2B is composed of three hydrophobic membrane domains (two at the N terminus and a single one at the C terminus), which have been shown to be crucial for the association of the protease with membranes within the CM/PC (Brinkworth et al., 1999; Droll et al., 2000).
While the mechanism by which NS2B facilitates protease activity is not completely understood, comparisons between the crystal structures of the NS2B/NS3 proteases of DEN2 and WNV with those of DEN2 NS3pro alone have revealed that NS3 undergoes substantial rearrangement in the presence of the NS2B cofactor (Murthy et al., 2000; Erbel et al., 2006; Aleshin et al., 2007). The hydrophilic cofactor domain, NS2B49–88 is shown in the crystal structure of WNV NS2B/NS3pro to form a belt that wraps around the NS3 protease domain (Fig. 1a). This is thought to constrain the flexibility of NS3 and force it to adopt the active conformation. The crystal structure also shows that the C terminus of this cofactor domain comes into close proximity with the bound aldehyde inhibitor, with NS2B83–87 forming part of the substrate-binding cleft and providing direct interactions, notably with the P2 lysine. This explains why, in the absence of NS2B, NS3pro is unable to interact properly with substrates extending beyond the S1 site and is thus inactive (Yusof et al., 2000; Erbel et al., 2006).
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) and the DEN NS2B/NS3pro (Erbel et al., 2006). While the N-terminal residues NS2B50–62 remain associated with the N-terminal β-barrel of NS3pro, the C-terminal residues NS2B63–93 extend into the solvent and do not form part of the C-terminal β-barrel. In inhibitor bound crystals these C-terminal residues form part of the substrate-binding cleft with residue NS2B84 interacting directly with P2 of the inhibitor.
Various site-direct mutagenesis studies have already been directed to residues within the NS2B cofactor domain of Yellow fever virus (Chambers et al., 1993, 2005; Droll et al., 2000), Alkhurma virus (Pastorino et al., 2006) and DEN2 (Niyomrattanakit et al., 2004). Each of these studies has selectively focused on particular residues within NS2B and, while some have been shown to be important for proteolytic activity, no study has empirically assessed the role of each residue within the central cofactor domain. For this reason we conducted an alanine mutagenesis screen throughout the entire cofactor domain of NS2B51–92.
Previously, we reported the expression and purification of a recombinant WNV protease, in which the cofactor domain of NS2B was linked to the proteolytic domain of NS3 by a nonapeptide linker (Nall et al., 2004). This protease was shown to be proteolytically active against hexapeptide (Nall et al., 2004) and tetrapeptide (Chappell et al., 2006) substrates containing a para-nitroanilide (pNA) group in the P1' position. In this study, residues within the cofactor domain (NS2B51–92) of this construct were individually mutated to alanine, excluding native alanine residues Ala58 and Ala66. Mutations were inserted by PCR amplification of the plasmid using partially overlapping primers containing the desired mutation. Template plasmid was digested with methylation-specific DpnI and competent Escherichia coli were transformed with the plasmid. Incorporation of the desired mutation was confirmed by sequence analysis. Expression and purification of mutated proteases were conducted as described previously (Nall et al., 2004) and protein purity was confirmed by 12 % SDS-PAGE (results not shown).
The activity of the mutated proteases was assessed against the tetrapeptide substrate Ac-LKKR-pNA, using an in vitro protease assay described previously (Nall et al., 2004). The activities of the mutated proteases compared with the control are shown in Fig. 1(b)
. The majority of mutations (36 of 40) resulted in some level of decrease in activity, with only mutations D51A, T57A, T69A and S72A retaining activity similar to the control recombinant protease. Two mutations were found to completely inactivate the protease, G83A and F85A. Two regions of the cofactor (NS2B59–62 and NS2B75–87), referred to as site 1 and 2, respectively, were identified where alanine mutagenesis greatly affected proteolytic activity, with the majority of substitutions decreasing activities below 20 %.
An alignment of cofactor domain sequences from selected mosquito-borne flaviviruses is shown in Fig. 2. The majority of the mutations that had a large effect on proteolytic activity mapped to sites of strong residue conservation, suggesting that they are likely to play similar important roles in the activation of other flavivirus NS3 proteases. Some of the residues identified in this study as important for protease activity have been noted previously. In DEN2, mutagenesis of Trp62, Leu75, Ile77 and Ile79 and in Alkhurma virus, the homologous mutagenesis of Trp60, Leu73 and Gln77 all produced large decreases in protease activity similar to those observed in our study (Niyomrattanakit et al., 2004; Pastorino et al., 2006). A charged region at the N-terminal end of this central hydrophilic domain of the Yellow fever virus NS2B, comprising residues Glu52, Lys54 and Lys55 has been identified as being important to protease activity; however, significant loss of protease activity only became apparent when two or three residues were simultaneously mutated to alanine, suggesting some redundancy of an overall charge effect (Droll et al., 2000).
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). Indeed, mutagenesis of T69A and S72A resulted in small increases in activity. The high level of conservation of NS2B69–72 may indicate a role other than in protease activation or one that is not assessed by the in vitro based assay system. It is possible, for example, that these residues are important in the context of the cell membrane anchored, full-length NS2B cofactor. A degree of flexibility within this region could potentially play a role in cofactor driven substrate association and dissociation post-cleavage. As described above, this region of the cofactor is disengaged from NS3pro in the absence of substrate (Erbel et al., 2006; Aleshin et al., 2007) with the highly conserved Trp62 acting as a form of anchor at the end of the N-terminal NS2B50–62 region, tethering the cofactor to NS3pro. The most highly conserved motif within this region, 70GxS72, would provide a degree of flexibility for this region, a flexibility that may also facilitate the binding of different sized residues into the substrate-binding cleft. Consistent with the flexibility of this region, proteolysis of a recombinant DEN2 NS2B/NS3 protease with endoprotease Asp-N produced cleavage within the cofactor domain at Asp81. Following proteolysis, NS2B50–80 remained tightly bound to NS3pro, while NS2B81–96 was no longer associated. Surprisingly, this truncated protease, containing only NS2B50–80 and NS36–179, retained 52 % of the original activity, suggesting that the substrate interactions supplied by the residues NS2B83–87, while important, are not critical for protease activity (Melino et al., 2006).
Analysis of the location of sites 1 and 2 (NS2B59–62 and NS2B75–87) within the crystal structure provides some clues as to their role in the activation of the NS3 protease. The regions are highlighted in Fig. 3 in the crystal structure of WNV NS2B/NS3pro (pdb 2FP7; Aleshin et al., 2007). Site 1 (NS2B59–62) is shown to bind to a region in the NS3pro N-terminal β-barrel with conserved residues Ile60 and Trp62 binding to adjacent hydrophobic pockets. These residues constitute the C-terminal end of the region of the cofactor NS2B50–62, which remains tightly bound in both the inhibitor-bound and substrate-free crystal structures (Aleshin et al., 2007; Erbel et al., 2006). Tight association of these residues is presumably required for the correct interaction between NS2B and NS3pro to prevent NS2B from disassociating from the substrate-free protease.
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We have previously demonstrated that Asn84 provides an important hydrogen bond with the substrate at P2, as its substitution for serine was able to alter substrate preference at P2 (Chappell et al., 2006). Therefore, it is likely that large decreases in proteolytic activity following mutation within NS2B75–87 are partially due to incorrect protein folding as well as decreased substrate-binding ability.
The critical cofactor-binding sites identified in this study are potential targets for allosteric inhibitor development. These inhibitors would act through the disruption or prevention of NS3pro association with NS2B. This novel antiviral strategy may circumvent some of the current problems facing the development of substrate-based competitive inhibitors, such as the highly charged nature of the interaction at the substrate-binding cleft.
Site 1 (NS2B59–62), in which the conserved residues Ile/Val60 and Trp62 have been shown to bind to adjacent pockets of NS3, could be targeted by small aromatic, drug-like compounds. Displacement of the cofactor from this region is likely to prevent correct folding of the protease and thereby lead to inactivation. However, because this region of the cofactor remains tightly associated in both inhibitor-bound and substrate/inhibitor-free crystal structures (Aleshin et al., 2007; Erbel et al., 2006), it will be difficult to develop compounds with high enough affinity to displace the bound cofactor.
Site 2 (NS2B75–87) forms a β-loop that binds to a deep pocket in close proximity to the active site. This region of the cofactor is believed to be more flexible and not associated with the substrate-free protease (Aleshin et al., 2007) and so may be more accessible to potential inhibitors. Due to the close proximity of this region to the active site, where it forms part of the substrate-binding cleft, it is likely that displacement of this region will interfere with substrate binding. It should be noted, however, that removal of NS2B81–93 by proteolysis does not completely inactivate the enzyme (Melino et al., 2006), and so it remains to be demonstrated that inhibitor targeting of this site will have a sufficiently profound effect on protease activity to abrogate viral replication.
In silico docking of virtual compound libraries into both of these potential inhibitor-binding sites is currently under way. These studies should lead to the identification of novel inhibitors and provide proof of concept for the use of cofactor based inhibitors against the flavivirus NS2B/NS3 protease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brinkworth, R. I., Fairlie, D. P., Leung, D. & Young, P. R. (1999). Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor. J Gen Virol 80, 1167–1177.[Abstract]
Chambers, T. J., Weir, R. C., Grakoui, A., McCourt, D. W., Bazan, J. F., Fletterick, R. J. & Rice, C. M. (1990). Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proc Natl Acad Sci U S A 87, 8898–8902.
Chambers, T. J., Nestorowicz, A., Amberg, S. M. & Rice, C. M. (1993). Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B–NS3 complex formation, and viral replication. J Virol 67, 6797–6807.
Chambers, T. J., Droll, D. A., Tang, Y., Liang, Y., Ganesh, V. K., Murthy, K. H. & Nickells, M. (2005). Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities. J Gen Virol 86, 1403–1413.
Chappell, K. J., Stoermer, M. J., Fairlie, D. P. & Young, P. R. (2006). West Nile virus NS3 protease: insights into substrate binding and processing through combined modelling, protease mutagenesis and kinetic studies. J Biol Chem 281, 38448–38458.
Droll, D. A., Krishna Murthy, H. M. & Chambers, T. J. (2000). Yellow fever virus NS2B–NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function. Virology 275, 335–347.[CrossRef][Medline]
Erbel, P., Schiering, N., D'Arcy, A., Renatus, M., Kroemer, M., Lim, S. P., Yin, Z., Keller, T. H., Vasudevan, S. G. & Hommel, U. (2006). Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 13, 372–373.[CrossRef][Medline]
Falgout, B., Miller, R. H. & Lai, C. J. (1993). Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B–NS3 protease activity. J Virol 67, 2034–2042.
Gorbalenya, A. E., Donchenko, A. P., Koonin, E. V. & Blinov, V. M. (1989). N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res 17, 3889–3897.
Gould, L. H. & Fikrig, E. (2004). West Nile virus: a growing concern? J Clin Invest 113, 1102–1107.[CrossRef][Medline]
Hayes, C. G. (2001). West Nile Virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci 951, 25–37.[Medline]
Komar, N. & Clark, G. G. (2006). West Nile virus activity in Latin America and the Caribbean. Rev Panam Salud Publica 19, 112–117.[Medline]
Melino, S., Fucito, S., Campagna, A., Wrubl, F., Gamarnik, A., Cicero, D. O. & Paci, M. (2006). The active essential CFNS3d protein complex. FEBS J 273, 3650–3662.[CrossRef][Medline]
Murthy, H. M., Judge, K., DeLucas, L. & Padmanabhan, R. (2000). Crystal structure of Dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. J Mol Biol 301, 759–767.[CrossRef][Medline]
Nall, T. A., Chappell, K. J., Stoermer, M. J., Fang, N. X., Tyndall, J. D., Young, P. R. & Fairlie, D. P. (2004). Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease. J Biol Chem 279, 48535–48542.
Niyomrattanakit, P., Winoyanuwattikun, P., Chanprapaph, S., Angsuthanasombat, C., Panyim, S. & Katzenmeier, G. (2004). Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J Virol 78, 13708–13716.
Pastorino, B. A., Peyrefitte, C. N., Grandadam, M., Thill, M. C., Tolou, H. J. & Bessaud, M. (2006). Mutagenesis analysis of the NS2B determinants of the Alkhurma virus NS2B–NS3 protease activation. J Gen Virol 87, 3279–3283.
van der Meulen, K. M., Pensaert, M. B. & Nauwynck, H. J. (2005). West Nile virus in the vertebrate world. Arch Virol 150, 637–657.[CrossRef][Medline]
Wengler, G. (1991). The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 184, 707–715.[CrossRef][Medline]
Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K. & Khromykh, A. A. (1997). Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J Virol 71, 6650–6661.[Abstract]
Yusof, R., Clum, S., Wetzel, M., Murthy, H. M. & Padmanabhan, R. (2000). Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J Biol Chem 275, 9963–9969.
Received 16 September 2007;
accepted 6 December 2007.
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