Short Communication Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B–NS3 proteinase Ilian Radichev,3 Sergey A. Shiryaev,3 Alexander E. Aleshin, Boris I. Ratnikov, Jeffrey W. Smith, Robert C. Liddington and Alex Y. Strongin Correspondence Alex Y. Strongin strongin@burnham.org Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA Received 7 August 2007 Accepted 20 November 2007 West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B–NS3pro structure, we hypothesized that the G 22 residue and the negatively charged patch D 32 DD 34 of NS2B were part of an important configuration required for NS2B–NS3pro activity. Our experimental data confirmed that G 22 and D 32 DD 34 substitution for S and AAA, respectively, inactivated NS2B–NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B–NS3pro. Because of the significant level of homology in flaviviral NS2B–NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro. West Nile virus (WNV), a member of the family Flaviviridae, is an enveloped, positive-strand, 11 kb RNA virus that is transmitted by mosquitoes (Mukhopadhyay et al., 2005; van der Meulen et al., 2005). WNV causes central nervous system damage unless specific treatment is administered (Madden, 2003; Wang et al., 2004). The genomic RNA of WNV encodes a polyprotein precursor which consists of three structural proteins (C, capsid; prM, membrane and E, envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3- NS4A-NS4B-NS5. Polyprotein processing by the host cell signal peptidase and furin in the endoplasmic reticulum, and also by the viral two-component NS2B–NS3 proteinase (NS2B–NS3pro) is required to generate individual viral proteins (Beasley, 2005; Cahour et al., 1992; Mukhopadhyay et al., 2005). Full-length NS3 represents a multifunctional protein in which the N-terminal 184 aa residues represent NS3pro and the C-terminal sequence codes for a helicase. NS3pro is responsible for the cleavage of the C protein and at the NS2A/NS2B, NS2B/NS3, NS3/ NS4A and NS4B/NS5 boundaries. In addition, the cleavage of NS4A by NS3pro is required (Lin et al., 1993) for the subsequent efficient cleavage of the NS4A/NS4B junction by the signal peptidase (Preugschat & Strauss, 1991). Inactivating mutations of the NS3pro cleavage sites in the polyprotein precursor abolished viral infectivity (Chambers et al., 1993, 2005). In many flavivirus species including WNV, NS2B functions as a cofactor and promotes the productive folding and the activity of NS3pro. The cofactor activity of the 48 aa central portion of NS2B is roughly equivalent to that of the entire NS2B sequence (Leung et al., 2001). Structural studies suggest that NS2B–NS3pro exhibits two alternative, productive and unproductive, conformations. In the productive conformation, NS2B wraps around NS3pro, completing, in a precise and well-defined fashion, the structure of the active site (Aleshin et al., 2007; Erbel et al., 2006). In agreement, the NS2B-free NS3pro enzyme is inactive (Falgout et al., 1991, 1993). These unique cofactor–protease domain interactions are common for the multiple flaviviruses (Bessaud et al., 2005; Droll et al., 2000; Wu et al., 2003). The requirement of these interactions for catalysis raises the possibility of designing allosteric inhibitors that interfere with the NS2B fold rather than directly targeting the catalytic triad of NS3pro. The inhibitor design, however, requires the precise knowledge of the functional determinants of the cofactor that are 3These authors contributed equally to this work. The sequences of the mutant primers are available with the online version of this paper. Journal of General Virology (2008), 89, 636–641 DOI 10.1099/vir.0.83359-0 636 0008-3359 G 2008 SGM Printed in Great Britain