Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus

Background: Chronic hepatitis resulting from the hepatitis C virus (HCV) in fection leads to cirrhosis in at least half the infected patients and incre ases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal c ascades caused by the interaction with viral antigens. Although a great amo unt of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, w e have demonstrated that the full-length non-structural protein 3 of HCV (N S3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-stru ctural protein 3 of hepatitis C virus as modulator of protein phosphorylati on mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a;144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphoryla tion mediated by cAMP-dependent protein kinase. fur J Biochem 1996;237:611- 618) associate to stable complexes with core histones H2B and H4. The chang es of the properties of histones as substrate for cAMP-dependent protein ki nase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. Objective: in the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. Study design: HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to his tones was biochemically characterized. The region of NS3 involved in the in teraction with histones was defined by proteolytic fragmentation, microsequ encing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the b inding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. Results: The investigated fragment of HCV polyprotein consisting of amino a cid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of hist one to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprote in. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. Conclusions: Our results provide the first evidence that NS3 binds and affe cts the functions of core histones. The mechanism by which the NS3 interfer es with the histone functions involves conformational changes of histone mo lecule. (C) 1999 Elsevier Science B.V. All rights reserved.