Mutagenesis of the West Nile virus NS2B cofactor domain reveals two regions essential for protease activity.

The flavivirus NS2B/NS3 protease has received considerable attention as a target for the development of antiviral compounds. While substrate based inhibitors have been the primary focus to date, an approach focussing on NS2B cofactor displacement could prove to be an effective alternative. To understand better the role of the NS2B cofactor in protease activation, we conducted an alanine mutagenesis screen throughout the 42-residue central cofactor domain (NS2B(51-92)) of West Nile virus (WNV). Two sites critical for proteolytic activity were identified (NS2B(59-62) and NS2B(75-87)), where the majority of substitutions were found to significantly decrease proteolytic activity of a recombinant WNV NS2B/NS3 protease. These findings provide mechanistic insights into the structural and functional role that the cofactor may play in the substrate-bound and free protease complexes as well as providing novel sites for targeting new antiviral inhibitors.

[1]  Zheng Yin,et al.  Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus , 2006, Nature Structural &Molecular Biology.

[2]  G. Clark,et al.  West Nile virus activity in Latin America and the Caribbean. , 2006, Revista panamericana de salud publica = Pan American journal of public health.

[3]  A. Gamarnik,et al.  The active essential CFNS3d protein complex , 2006, The FEBS journal.

[4]  C. Hayes West Nile Virus: Uganda, 1937, to New York City, 1999 , 2001, Annals of the New York Academy of Sciences.

[5]  K. Murthy,et al.  Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities. , 2005, The Journal of general virology.

[6]  R. Yusof,et al.  Purified NS2B/NS3 Serine Protease of Dengue Virus Type 2 Exhibits Cofactor NS2B Dependence for Cleavage of Substrates with Dibasic Amino Acids in Vitro* , 2000, The Journal of Biological Chemistry.

[7]  G. Wengler,et al.  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. , 1991, Virology.

[8]  L. DeLucas,et al.  Crystal structure of Dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. , 2000, Journal of molecular biology.

[9]  Martin J. Stoermer,et al.  Enzymatic Characterization and Homology Model of a Catalytically Active Recombinant West Nile Virus NS3 Protease* , 2004, Journal of Biological Chemistry.

[10]  R. Miller,et al.  Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity , 1993, Journal of virology.

[11]  T. Chambers,et al.  Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function. , 2000, Virology.

[12]  J. Mackenzie,et al.  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 , 1997, Journal of virology.

[13]  WEST NILE VIRUS NS 3 PROTEASE : INSIGHTS INTO SUBSTRATE BINDING AND PROCESSING THROUGH COMBINED MODELLING , PROTEASE MUTAGENESIS AND KINETIC STUDIES , 2006 .

[14]  Alex Y Strongin,et al.  Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold , 2007, Protein science : a publication of the Protein Society.

[15]  C. Peyrefitte,et al.  Mutagenesis analysis of the NS2B determinants of the Alkhurma virus NS2B-NS3 protease activation. , 2006, The Journal of general virology.

[16]  E. Fikrig,et al.  West Nile virus: a growing concern? , 2004, The Journal of clinical investigation.

[17]  P. Niyomrattanakit,et al.  Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation , 2004, Journal of Virology.

[18]  R J Fletterick,et al.  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. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[19]  V. Blinov,et al.  N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. , 1989, Nucleic acids research.

[20]  D. Fairlie,et al.  Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor. , 1999, The Journal of general virology.

[21]  C. Rice,et al.  Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication , 1993, Journal of virology.

[22]  Martin J. Stoermer,et al.  Insights to Substrate Binding and Processing by West Nile Virus NS3 Protease through Combined Modeling, Protease Mutagenesis, and Kinetic Studies* , 2006, Journal of Biological Chemistry.

[23]  H. Nauwynck,et al.  West Nile virus in the vertebrate world , 2005, Archives of Virology.