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

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases. The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases. West Nile virus (WNV) 1The abbreviations used are: WNV, West Nile virus; pNA, p-nitroanilide; HCV, hepatitis C virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Rt, retention time. is a member of the Flavivirus genus and is transmitted by mosquitoes, primarily Culex species (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar), between avian reservoir hosts and vertebrate dead-end hosts including humans and horses. Many viruses within this genus are medically important pathogens, including dengue, Japanese encephalitis, tick-borne encephalitis, and yellow fever viruses. WNV was first isolated in 1937 in Uganda's West Nile province and was subsequently found in regions of Africa, the Middle East, Europe, Russia, South-western Asia, and Australia (less severe subtype, Kunjin) (2Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar). Human infection is generally asymptomatic or causes a mild febrile disease, West Nile fever (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar). However, in a small number of cases, predominantly in the elderly, the infection with WNV results in encephalitis or meningitis that can be fatal (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar). Over the last decade, there has been an increase in the frequency of human outbreaks and severity of disease with recent epidemics in Israel (1998), Romania (1999), Russia (1999), and New York (1999) (3Lanciotti R.S. Roehrig J.T. Deubel V. Smith J. Parker M. Steele K. Crise B. Volpe K.E. Crabtree M.B. Scherret J.H. Hall R.A. MacKenzie J.S. Cropp C.B. Panigrahy B. Ostlund E. Schmitt B. Malkinson M. Banet C. Weissman J. Komar N. Savage H.M. Stone W. McNamara T. Gubler D.J. Science. 1999; 286: 2333-2337Crossref PubMed Scopus (1283) Google Scholar). Since the introduction of WNV into New York in 1999, it has spread rapidly throughout the United States (4,156 infections and 284 deaths in 44 states in 2002, 9862 infections and 264 deaths in 2003) (reported in 2000 by the CDC, Division of Vector-Borne Infectious Disease: West Nile Virus, Center for Disease Control and Prevention, www.cdc.gov/ncidod/dvbid/westnile/index.htm), Canada, and Mexico and has recently appeared in the United Kingdom (5Buckley A. Dawson A. Moss S.R. Hinsley S.A. Bellamy P.E. Gould E.A. J. Gen. Virol. 2003; 84: 2807-2817Crossref PubMed Scopus (178) Google Scholar). There is currently no vaccine or antiviral treatment available for human WNV infection. However, a chimeric live vaccine is in clinical trials and a veterinary vaccine is licensed for use in equines and exotic zoo birds (6Tesh R.B. Arroyo J. Travassos Da Rosa A.P. Guzman H. Xiao S.Y. Monath T.P. Emerg. Infect. Dis. 2002; 8: 1392-1397Crossref PubMed Scopus (124) Google Scholar). Flaviviruses are small enveloped viruses containing a single-stranded positive sense RNA genome of 10–11 kb with a single large open reading frame encoding a polyprotein precursor of ∼3400 amino acids. Gene expression requires both host and a virally encoded protease to process the polyprotein precursor into the individual functional proteins. They comprise three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (7Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1596) Google Scholar). The viral protease encoded within the N-terminal third of NS3 is responsible for cleavage at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 gene junctions and also at a site near the C terminus of the C protein to promote efficient generation of prM (Fig. 1) (8Chambers T.J. Weir R.C. Grakoui A. McCourt D.W. Bazan J.F. Fletterick R.J. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8898-8902Crossref PubMed Scopus (287) Google Scholar, 9Lobigs M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6218-6222Crossref PubMed Scopus (123) Google Scholar). Mutation of residues at any of these cleavage sites in the related yellow fever virus was shown to prevent efficient cleavage and abolished virus infectivity in cell culture (10Amberg S.M. Rice C.M. J. Virol. 1999; 73: 8083-8094Crossref PubMed Google Scholar, 11Lin C. Chambers T.J. Rice C.M. Virology. 1993; 192: 596-604Crossref PubMed Scopus (38) Google Scholar, 12Nestorowicz A. Chambers T.J. Rice C.M. Virology. 1994; 199: 114-123Crossref PubMed Scopus (70) Google Scholar, 13Chambers T.J. Nestorowicz A. Rice C.M. J. Virol. 1995; 69: 1600-1605Crossref PubMed Google Scholar), highlighting a vital role for the NS3 protease in replication. The essential nature of this protease in the virus life cycle is the basis for interest in NS3 as a possible target for developing antiviral inhibitors. NS3 is a multifunctional protein in which the N-terminal 184 amino acids encode for the protease and the C-terminal region encodes a nucleotide triphosphatase, an RNA triphosphatase, and a helicase (14Gorbalenya A.E. Donchenko A.P. Koonin E.V. Blinov V.M. Nucleic Acids Res. 1989; 17: 3889-3897Crossref PubMed Scopus (204) Google Scholar, 15Wengler G. Virology. 1991; 184: 707-715Crossref PubMed Scopus (162) Google Scholar, 16Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar). The NS3 protease is a trypsin-like serine protease with a classic catalytic triad (His-51, Asp-75, and Ser-135) (17Bazan J.F. Fletterick R.J. Virology. 1989; 171: 637-639Crossref PubMed Scopus (250) Google Scholar). Protease activity has been shown for a number of related flaviviruses to be dependent on the association of NS2B as a cofactor (18Falgout B. Pethel M. Zhang Y.M. Lai C.J. J. Virol. 1991; 65: 2467-2475Crossref PubMed Google Scholar). A central 40 amino acid hydrophilic domain within the largely hydrophobic NS2B protein has been shown to be sufficient for cofactor activity (19Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 20Falgout B. Miller R.H. Lai C.J. J. Virol. 1993; 67: 2034-2042Crossref PubMed Google Scholar). The flanking hydrophobic domains within NS2B are likely to function in promoting membrane association of NS2B-NS3 (21Clum S. Ebner K.E. Padmanabhan R. J. Biol. Chem. 1997; 272: 30715-30723Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 22Brinkworth R.I. Fairlie D.P. Leung D. Young P.R. J. Gen. Virol. 1999; 80: 1167-1177Crossref PubMed Scopus (89) Google Scholar). NS2B-NS3pro has high specificity for substrate processing requiring a dibasic recognition sequence (P2-Lys, P1-Arg) that is conserved throughout the flaviruses (7Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1596) Google Scholar). This relatively unusual substrate specificity suggests that inhibitors could be somewhat selective over most host serine proteases (19Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Fundamental to the design of a specific inhibitor is a detailed understanding of the interactions between protease residues in the active site and substrates. The structure of the WNV NS3 protease is unknown, but there are reported crystal structures for related NS3 proteases of hepatitis C virus (HCV) with a cofactor (23Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and Dengue-2 virus without NS2B cofactor (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). the structure was of an this a structure of the WNV NS3 sequence with the and Dengue-2 virus NS3 protease was used to residues in the S1 and S2 that important interactions with substrate residues T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues in a recombinant active by and for the mutant WNV proteases and amino acids and and and of the other of by high on a a of and high was on a with a and a a by high over and The of the was by on a on containing in on a at K. by of and of the K. of and Nucleic New Google Scholar). of the on or C. B. B. Scholar). Homology Nile virus NS3 protease the structures of Dengue-2 virus NS3 protease without cofactor (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and with inhibitor H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar) and hepatitis C virus NS3 protease with cofactor (23Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). the and Homology within R. N. D.W. Rice J. Chem. PubMed Scopus Google Scholar) on a and T.J. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar). structure predictions the for of the three NS3 protease J. Biol. 1999; Scholar). potential was on the WNV NS3 protease the in E. H. 2003; PubMed Scopus Google Scholar). A substrate on the cleavage site was into the active site of a of the WNV NS3 protease G. R.C. J. Biol. 1995; PubMed Scopus Google Scholar). A between the substrate Arg and WNV protease was used on understanding of interactions D.P. R.C. Abbenante G. M.J. J. Chem. PubMed Scopus Google Scholar, D. Abbenante G. Fairlie D.P. J. Chem. PubMed Scopus Google Scholar). J. D. and D. Chem. in structures for G. R.C. J. Biol. 1995; PubMed Scopus Google Scholar). expression WNV T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) WNV was used as a for mutant S135A, S160A, and S163A) in WNV NS3 the first of with the WNV and WNV to by the the in the of the WNV and WNV The with and and into The of the mutations was by sequence used for of WNV and in a and was used for high expression of N-terminal recombinant proteins. of the with the expression in of containing and at the The expression of the recombinant protein was by the of to a of and for an at by and at protein cell in and protease inhibitors in an to cleavage of recombinant found that these serine protease inhibitors WNV T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by three a and The recombinant with a N-terminal by on a that been was with the of cell and at on a for to the protein to to the The was and with of containing and the proteins into with containing The and and and the by substrate to the WNV NS3 protease cleavage site was by a reported G. Leung D. T. Fairlie D.P. Sci. 2001; Scholar) was and The are as and High was for found found recombinant WNV protease and S135A, S160A, and S163A) a hexapeptide substrate to of the cleavage site with a in the The cleavage of the the substrate by the WNV proteases a at protease activity to be The was in a with a of containing recombinant protease and and with processing by T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of a of of protease and substrate in was by substrate with by The was at for in a and the in was in and a containing no was also proteases that found to activity the in substrate to for of for the which activity the it was in at the of and substrate to of the as a function of the substrate The that for The are reported as the means Protease and of structural of the WNV T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was on the sequence and the crystal structures of Dengue-2 virus in with a inhibitor H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar) and of in with cofactor (Fig. J.L. C. T. J.A. Chambers W. Rice C.M. M.A. P.R. J.A. 87: Full Text Full Text PDF PubMed Scopus Google Scholar). The structure was to the modeling of the WNV in the of NS2B The of WNV is shown in highlighting and hydrophobic regions with a reported substrate to the WNV NS4B/NS5 cleavage in the active on with the cofactor the protease with the region in the of of the The active site is in or on a largely hydrophobic region of the by regions at and in The that the S1 site was the that the S2 and and that the substrate on the of the protease (Fig. Asp-129, Tyr-150, Ser-160, and Ser-163 are to the S1 Asn-152 is to be a key in the S2 (Fig. C and and these for The S1 was the of study as it to be the most site for with a and Ser-163 are to residues in the S1 of the Dengue-2 virus NS3 protease that been to with P1 of substrate (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar), and has been by as important for the activity of Dengue-2 virus protease activity B. J. Virol. 1998; PubMed Google Scholar). to the and mutant WNV NS3 proteases to a interaction with the P1 Arg is for substrate recognition and The and to the possible interaction of these residues in S1 with Arg at P1 of the is conserved the flaviviruses and to of a that at the of the substrate and an with the P1 Lys or Arg of the cleavage site C.S. A. S. Fletterick R.J. PubMed Scopus Google Scholar). However, as this in the flavivirus NS3 protease with at the of the S1 site high of conservation the appeared to that could be relatively with retention of activity B. J. Virol. 1998; PubMed Google Scholar). for the Dengue-2 virus NS3 that to with the P1 Lys but to with a P1 Arg in of H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar). to the of for substrate processing by three mutant WNV and in and but of with of and at S1 a is important for interaction with Arg at P1 of the The mutations of and to the of and the of the S1 The these mutations was that could the available for the of a to the was to the of the of the Because of the dibasic substrate specificity of WNV NS3 also to the mutations at the S2 site in the The of the structure that Asn-152 was most likely to to the P2 Lys of the the to this protease was by of the catalytic serine to as a of these mutant proteases and the of the role of the residues in substrate to the predictions the structure and of WNV and recombinant WNV protease used in this study the essential 40 amino acid cofactor domain of NS2B to the 184 amino acid protease domain of NS3 by a as WNV T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This was into the expression and used to E. The recombinant WNV was by and was shown to be active on the cleavage sites of WNV T. Stoermer M.J. Fang Young P.R. Fairlie D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutations (Fig. into the WNV by and the mutant into the expression and into by sequence of the WNV and mutant by The species for of the a at and was to the active recombinant at and to the of in the of the catalytic mutant However, these in the WNV recombinant and mutant and the of these cleavage in the may an of catalytic activity and that the single amino acid and are active to the S135A, and are or to of WNV and the activities of the WNV and recombinant in the substrate on the sequence of the WNV NS4B/NS5 a single substrate of was used and the in the substrate cleavage of the mutant with the WNV and also a containing no The and selected for a of substrate The and catalytic activity to that of the and the catalytic and as for WNV and with or activity by at substrate for the recombinant which activity the the was at a substrate for WNV and are shown in for substrate processing by of in a of containing and and in at an of and to was in at the of and to in a The between the used to provide for the role of the residues in substrate The of a but a in catalytic to the of substrate increase in The of an large in catalytic in of a in and also a large in substrate increase in The an large in catalytic in of a in and a in substrate increase in The and in a in catalytic to a in and a in substrate increase in The a in catalytic to a in and a small in substrate increase in a in catalytic of a in and a small in substrate increase in of to of on protease is used to provide for the of individual residues in substrate and of protease residues structure and to be important for interactions with P1 and P2 residues of the dibasic substrate to be is conserved throughout flavivirus NS3 proteases. at the of the S1 in the of WNV as in Dengue-2 virus by (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar). The of is to a interaction with the Arg at P1 of the of the by in an with results for Dengue-2 virus B. J. Virol. 1998; PubMed Google Scholar) and the that makes a interaction with the P1 The of that the the catalytic activity but a in catalytic and a small in substrate increase in This the interaction that requires the of both of these S1 The in catalytic might be to the of a between the and P1 of the substrate or to an increase in the of the is also conserved throughout the flavivirus NS3 proteases. The Ser-163 to the S1 and this has been to an interaction with a P1 Arg in the crystal structure of the inhibitor to Dengue-2 virus H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar). Mutation of Ser-163 to the that Ser-163 makes a interaction with the P1 a is also conserved throughout flavivirus NS3 proteases. on to of was to at the of the S1 and the interaction with the substrate P1 (17Bazan J.F. Fletterick R.J. Virology. 1989; 171: 637-639Crossref PubMed Scopus (250) Google Scholar). of the Dengue-2 virus and to that at the of the S1 protease The of the substrate interaction by was also in which that in Dengue-2 virus NS3 a on protease activity B. J. Virol. 1998; PubMed Google Scholar). to the B. J. Virol. 1998; PubMed Google Scholar), in this study that the protease activity in vitro. provide an important interaction with the a or with the positive of the P1 for Dengue-2 virus with inhibitor that P1 Arg in it is to and it is to and Ser-163 H.M. K. Padmanabhan R. J. Biol. PubMed Scopus Google Scholar). this is may a role in the of the P1 Arg it into the S1 it can subsequently to and Ser-163 The for the mutant are with this the large the that is in substrate The in a in catalytic a small in substrate increase in and a large in Because is the increase in in the catalytic substrate This that to and Ser-163 without first with the substrate in efficient an interaction that is in the of the P1 Arg into the S1 the may the P1 Arg into with and Ser-163 but it in a which in The mutant also catalytic and a small in substrate increase in this mutant or This of an acid by an to the that a interaction is important between and the P1 Arg of but is as for as or is by a interaction as in the of the of the a in protease the activity of in this study to be to the (Fig. to an B. J. Virol. 1998; PubMed Google Scholar) for by Dengue-2 virus NS2B-NS3pro may be the of of the residues protease this of catalytic may be relevant the cleavage of an the cleavage of a the sequence may to the association of substrate and the protease active provide an interaction with the P1 Arg as this may be in the design of inhibitors. an inhibitor to interactions with Tyr-150, and Asp-129, the be that of the P1 Arg and provide a was shown by the of WNV to the S2 and is likely to an interaction with the P2 The of Asn-152 to the the that it an essential interaction with the P2 Lys a with the of interactions with both P1 and P2 substrate cleavage was of any of the mutant containing or that the S1 was to provide a substrate interaction but was in this study to the of the S1 with the a in catalytic of a in substrate increase in This may be the of a small increase in the of the S1 for the P1 Arg which is of a large for the protease most the S1 small to of the P1 is conserved in flavivirus NS3 but the of WNV that may an interaction to the P1 The in a in catalytic and a in substrate increase in that may provide a interaction with the a This a in substrate recognition between of the NS3 protease This study important in the WNV NS2B/NS3 protease for recognition of a dibasic substrate has that the residues at both S1 and S2 are essential for substrate recognition and catalytic efficiency.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.