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.

The binding of one ADP molecule at the catalytic site of the nucleotide depleted IF,-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F,-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The halftime of this process is about 30 s, and the inhibition is correlated with P, dissociation from the F,-ATPase catalytic site (uni-site catalysis). The F,-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (T,,~ 20 s) or(ii) binding of P, in addition to MgADP and the formation of the triple F,-ATPase-MgADP-P, complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F,-ATPase. It is proposed that ATP-dependent dissociation of the F,-ATPase-GDP complex occurs more rapidly, than that of the F,-ATPase-ADP complex.

Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.