Cleavage preference distinguishes the two-component NS2B–NS3 serine proteinases of Dengue and West Nile viruses

Abstract

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

“…WNV NS2B/3 has been shown to preferentially cleave sites with a K at P2, whereas DENV NS2B/3 most efficiently cleaves sites with an R at this position. In contrast, WNV and DENV NS2B/3 proteases both preferentially cleave sites with an R over a K at the P1 position (25,33,62). These studies have indicated that WNV NS2B/3 has a strict preference for G at both the P1= and P2= positions, while DENV NS2B/3 is less selective and can cleave substrates with almost any amino acid at P1= and P2= (33).…”

“…In contrast, cleavage of the WNV C protein was efficient with any of the three residues at the P1= position. This fine specificity was not predicted by studies with recombinant NS2B/3 protease and short peptide substrates (33). We found that the sensitivity of WNV NS2B/3 to changes at the P1= position could be altered when residues at the P6 to P2 and P2= to P4= positions were mutated in various combinations.…”

“…Alignments of flavivirus NS2B/3 cleavage sites reveal a highly conserved dibasic motif at the P1 and P2 positions (upstream of the cleavage site) and a small-side-chain amino acid (G, A, S, or T) at the P1= position (downstream of the cleavage site), according to the Schechter and Berger nomenclature (24). This is reflected in the results of substrate specificity profiling studies performed with recombinant proteases of several flaviviruses, including WNV and DENV (25)(26)(27)(28)(29)(30)(31)(32)(33). However, while considerable insight has been gained from studies with recombinant proteases or model substrates, it is not known how the conditions used in these assays influence cleavage specificity or if they recapitulate the activity of NS2B/3 in infected cells.…”

“…Most studies to define protease cleavage site specificity employ short (3-to 8-amino-acid) synthetic peptide substrates and covalently linked and truncated forms of NS2B and NS3 (25)(26)(27)(28)(29)(30)(31)(32)(33). Furthermore, these experiments are performed in the absence of membranes, at basic pH (ϳpH 9), and in the presence of detergents and high concentrations of glycerol.…”

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

“…55 The used peptides were deduced from the cleavage sites in the immature WNV polyprotein and are summarized in Table 1.1. showing also the cleavage efficacies in percentage. (73) These data showed the preference of the NS2B-NS3 protease for substrates containing arginine as P1 residue, whereby one sequence with lysine in the NS2A protein was also accepted.…”