Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions

Chappell, Keith J.; Nall, Tessa; Stoermer, Martin J.; Fang, Ning; Tyndall, Joel D. A.; Fairlie, David P.; Young, Paul R.

doi:10.1074/jbc.m409931200

Cited by 61 publications

(68 citation statements)

References 37 publications

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“…Our results support and extend the previous findings of several groups (6,7,10,19,20,23,24,26,36) and also provide a structural rationale for the reduced selectivity of the DV proteinase, which tolerates well the presence of a number of amino acid residue types at both P1Ј and P2Ј positions. Importantly, earlier studies did not determine any significant difference in the P1Ј or P2Ј specificity between the WNV and DV enzymes (6,7,20).…”

Section: Discussionsupporting

confidence: 81%

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Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Shiryaev

Ratnikov

Aleshin

et al. 2007

J Virol

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The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1-P2 positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1 or P2. Structure-based modeling suggests that Arg 76 and Pro 131 -Thr 132 limit the P1-P2 subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu 76 and Lys 131 -Pro 132 widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3-P4 positions were fixed and the P1 and P2 positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.Dengue and West Nile viruses (DV and WNV) are members of the flavivirus genus within the Flaviviridae family. Four distinct, but structurally similar, serotypes represent DV (13). Flaviviruses are transmitted to animals, including humans, by either mosquito or tick bites. Flaviviruses are encoded by a single-strand, positive-sense, 11-kb RNA genome, which serves as an mRNA for protein synthesis and as a template for RNA replication in the host cell. There are three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) encoded by the viral genome (21). Proteinases of the host (furin and secretase) and of the virus [NS3 serine (pro)teinase (NS3pro)] are required for the complete processing of the polyprotein precursor and its transformation into mature viral proteins (4,27,31,34). Full-length NS3 is a bifunctional protein: its N-terminal and C-terminal portions encode the NS3pro domain and the helicase (NS3hel) domain, respectively (33, 35). NS3pro is responsible for cleavage of the capsid protein C, as well as the boundaries between NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/ NS4B, and NS4B/NS5 (6, 24).NS2B is an essential cofactor of NS3pro, and it is located immediately upstream in the polyprotein precursor (5, 9, 25, 26). The NS2B sequence includes three to four transmembrane helices that anchor the NS2B-NS3 heterodimer to the endoplasmic reticulum membrane. In vitro, the cofactor activity of the 35-to 48-residue central portion of NS2B is approximately equivalent to that of the entire NS2B sequence (11,12,19). Mutations in the NS3pro cleavage motifs in the polyprotein precursor abolish viral infectivity (2). These characteris...

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Section: Discussionsupporting

confidence: 81%

“…Full-length NS3 is a bifunctional protein: its N-terminal and C-terminal portions encode the NS3pro domain and the helicase (NS3hel) domain, respectively (33,35). NS3pro is responsible for cleavage of the capsid protein C, as well as the boundaries between NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/ NS4B, and NS4B/NS5 (6,24).…”

mentioning

confidence: 99%

Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Shiryaev

Ratnikov

Aleshin

et al. 2007

J Virol

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“…2A). Accordingly, in WNV or DV proteases, mutation of Asp129 to Glu (which may be too long) or Ala (which cannot saltbridge) abrogates catalysis (Chappell et al 2005). In trypsin, Asp189 plays an analogous role (Perona and Craik 1995;Hedstrom 2002), consistent with its similar P1 specificity.…”

Section: Substrate Specificity Of the Flavivirusesmentioning

confidence: 79%

Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Aleshin¹,

Shiryaev²,

Strongin³

et al. 2007

Protein Science

202

335

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Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an ''induced fit'' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.Keywords: protein structure/folding; conformational changes; enzymes; active sites; viral proteases; crystallography; enzyme inhibitors; mechanism-enzymes; protein structures-new Supplemental material: see www.proteinscience.org Flaviviruses, such as the closely related West Nile (WNV) and Dengue (DV) viruses, are members of a larger family called the Flaviviridae (Rice 1996; Burke and Monath 2001), which include the more distantly related hepaciviruses (e.g., Hepatitis C Virus [HCV]) and pestiviruses (e.g., Bovine virus diarrhea). WNV, DV, and HCV are recognized as major health threats that affect millions of people worldwide, with no specific countermeasures to treat them. WNV is transmitted to animals, including humans, by mosquito bites. It is encoded by a singlestrand, positive-sense, 11-kb RNA genome, which serves as mRNA for synthesis of the polyprotein precursor and as a template for genome replication in the host cell. The genome encodes three structural proteins found in the mature virion (C, prM, and E) and seven ''nonstructural'' (i.e., not part of the virion architecture) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) ( Abbreviations: NS, nonstructural; NS3pro, NS3 protease domain; WNV, West Nile Virus; DV, Dengue Virus; HCV, Hepatitis C Virus; JEV, Japanese Encephalitis Virus; KV, Kunjin Virus; YFV, Yellow Fever Virus; TM, transmembrane (helix); RSMD, root mean square deviation; NSLS, Brookhaven National Synchrotron Light Source; ALS, Berkeley Advanced Light Source; MAD, multiwavelength anomalous dispersion; DTT, dithiothreitol.Article published online ahead of print. Article and publication date are at

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“…Previous studies showed that the presence of the 48 residue central NS2B domain linked to the N-terminus of NS3 significantly enhanced the accumulation of soluble recombinant NS3pro in E.coli [18,20,23]. We used a similar approach to express the WNV NS2B-NS3pro.…”

Section: Resultsmentioning

confidence: 99%

Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B–NS3 junction region

Shiryaev

Aleshin

Ratnikov

et al. 2007

Protein Expression and Purification

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Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.

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Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions

Cited by 61 publications

References 37 publications

Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B–NS3 junction region

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