NS3 Peptide, a novel potent hepatitis C virus NS3 helicase inhibitor: its mechanism of action and antiviral activity in the replicon system.

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ((1)H and (15)N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.

L-arginine influences the structure and function of arginase mRNA in Aspergillus nidulans.

Expression of the arginase structural gene (agaA) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. Arginase mRNA has a long 5'-UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3' splicing sites. In this report we present evidence that L-arginine (i) binds directly to the arginase 5'-UTR; (ii) invokes drastic changes in the secondary structure of the 5'-UTR, unlike several other L-amino acids and D-arginine; and (iii) forces the selection of one of two 3' splice sites of an intron present in the 5'-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5'-UTR intron.