The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity.

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50 = 0.7 µM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

Synthesis and biological activity of 1H-benzotriazole and 1H-benzimidazole analogues--inhibitors of the NTpase/helicase of HCV and of some related Flaviviridae.

To improve anti-helical activity of analogues of 1H-benzotriazole and 1H-benzimidazole their N-alkyl derivatives were synthesized and tested for antihelicase activity against enzymes of selected Flaviviridae including hepatitis C virus (HCV), West Nile virus (WNV), Dengue virus (DENV) and Japanese encephalitis virus (JEV). 1- and 2-alkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole were obtained by direct alkylation of 4,5,6,7-tetrabromo-1H-benzotriazole with the use of respective alkyl halides in the presence of KOH in methanol, to give a mixture of 1- and 2- isomers, which was separated by flash column chromatography in good yield. The proportion of isomers strongly depended on the reaction time and temperature. 1- and 2-hydroxyethyl and 1- and 2-chloroethyl derivatives of the tetrabromobenzo-triazole were synthesized with the use of 2-bromoethanol and 1-bromo-2-chloroethane respectively as alkylating agents. N-alkylation of this benzotriazole compound enhanced inhibitory activity and selectivity towards the helicase activity of HCV NTPase/helicase. The most active were the 2-methyl, 2-ethyl and 2-propyl derivatives (IC50 approximately 6.5 microM in the presence of DNA as a substrate). Derivatives of the benzotriazole in which hydroxyethyl or chloroethyl replaced the alkyl substituents lost their inhibitory activity. Brominated or methylated benzotriazole N(1) ribosides also did not exert helicase inhibitory activity. Although a number of N(1) and N(2) alkyl derivatives exerted good HCV and WNV helicase inhibitory activity when DNA was used as substrate, the activity was strongly decreased or even disappeared when RNA was used as substrate. The cytotoxicity tests in Vero and HeLa Tat cells showed a substantial decrease of cytotoxicity of N-alkyl derivatives as compared to the parent benzotriazole.

Highly specific detection of antibodies to tick-borne encephalitis (TBE) virus in humans using a domain III antigen and a sensitive immune complex (IC) ELISA.

BACKGROUND: In contrast to most antibodies directed to the envelope glycoprotein of flaviviruses, those to the domain III (ED3) show serotype-specific reactions. OBJECTIVES: Only few epitopes are located on the ED3 [Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, et al. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005;11:522-30; Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 2005;437:764-9; Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile virus. J Virol 2006;80:6982-92], and highly sensitive assays may be required to detect the small number of human antibodies to this domain. STUDY DESIGN: We have used a sensitive immune complex (IC) ELISA to detect antibodies to the ED3 of TBE virus [Ludolfs D, Niedrig M, Paweska JT, Schmitz H. Reverse ELISA for the detection of anti-West Nile virus IgG antibodies in humans. Eur J Clin Microbiol Infect Dis 2007;26:467-73; Emmerich P, Gunther S, Schmitz H. Strain-specific antibody response to Lassa virus in the local population of west Africa. J Clin Virol 2008;42:40-4]. This assay was compared with two indirect ELISAs using either the ED3 (ED3 ELISA) or whole tissue culture virus (TCV) (TCV ELISA) as source of antigen. Sera of 45 patients with acute TBE infection and of 65 vaccinees were applied to determine the sensitivity of the IC ELISA. RESULTS: The IC ELISA detected antibodies in 107 out of 110 samples of TBE patients and vaccinees, 106 of which were also positive with the TCV ELISA. Both tests had a sensitivity of >or=96%. In contrast, the ED3 ELISA had a sensitivity of only 70%. Using samples of 98 West Africans and of 70 Europeans without any contact to TBE virus or TBE virus antigens, the specificity of the IC ELISA was 100% while the specificity of the commercial TCV ELISA varied between 30% with samples of people with acute dengue fever or with yellow fever vaccination and 100% with samples of students from Hamburg without any previous contact to TBE. CONCLUSION: Obviously, the IC ELISA is able to detect human antibodies to small antigens with only few serotype-specific epitopes with high specificity and sensitivity.

Regulation of the biochemical function of motif VI of HCV NTPase/helicase by the conserved Phe-loop.

In previous works, we demonstrated a potent inhibition of diverse protein kinase C (PKC) functions by a fragment of nonstructural protein 3 (NS3) of hepatitis C virus (HCV), mainly mediated by the Arg-rich amino acid motif HCV(1487-1500). This sequence is localized on the surface of Domain 2 of the NS3 NTPase/helicase in direct vicinity to a flexible loop that is localized between Val1458 and Thr1476. Here, we assessed the regulation of the accessibility of the Arg-rich amino acid motif for PKC by this loop, using two variants of domain 2. The first construct, termed NS3d2Delta, comprises the complete domain, HCV(1361-1503), devoid the loop. The second variant, NS3d2wt corresponds to wild type domain 2. The results indicated an enhanced inhibitory potential of NS3d2Delta towards rat brain PKC and towards the majority of PKC isoforms. This effect and the accompanying change of the mode of inhibition from a mixed mode, exerted by NS3d2wt to a competitive mode, exerted by NS3d2Delta are caused by the deletion of the loop. Accordingly, the presence of the intact loop abolished the binding of domain 2 to the tailed duplex RNA used as helicase substrate, without affecting the binding of dsDNA. Furthermore, a direct competition of dsRNA and PKC for the same binding site HCV(1487-1500), could be documented. The binding of dsRNA to NS3d2Delta previously overlaid with PPKCalpha was reduced to 30% and completely abolished in case of NS3d2Delta overlaid with cAMP-dependent protein kinase A (PKA).