The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Abstract

Summary. -hepatitis c virus (hcv) core (c) protein is thought to bind to viral rnA before it undergoes oligomerization leading to rnA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal c protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site (IreS). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral rnA by an IreS-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the c protein to bind to viral rnA. Serially truncated c proteins with deletions from 10 up to 45 n-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral rnA by a gel shift assay. The results showed that truncation of the c protein from its n-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the n-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGe. truncation by more than 20 amino acids resulted in a complete loss of ability of tagged c protein to bind to viral rnA. These results provide clues to the early events in the c protein -rnA interactions leading to c protein oligomerization, rnA encapsidation and virion assembly.Keywords: hepatitis c virus; core protein; rnA binding e-mail: sc2@nibmg.ac.in; phone: +91-33-25892151(256). Abbreviations: AM = anomalous migration; ArF = alternate reading frame; c protein = core protein; c124 = n terminal 124 amino acids of c protein; GeMSA = gel electrophoresis mobility shift assay; hcv = hepatitis c virus; IreS = internal ribosome entry site; Iec = ion exchange chromatography; nlP = nucleocapsidlike particles; MAb = monoclonal antibody; orF = open reading frame; Sec = size exclusion chromatography