tailieunhanh - Báo cáo khoa học: Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3¢-end of virus minus-strand RNA

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3¢-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3¢-end of the minus-strand HCV RNA in the presence of viral polymerase. | iFEBS Journal Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3 -end of virus minus-strand RNA Thérèse Astier-Gin Pantxika Bellecave Simon Litvak and Michel Ventura UMR-5097 CNRS Université Victor Segalen Bordeaux 2 Bordeaux France Keywords HCV minus strand RNA RdRp Correspondence Therese Astier-Gin CNRS UMR5097 Universite Victor Segalen Bordeaux 2 146 rue Leo Saignat 33076 Bordeaux cedex France Fax 33 5 57571766 Tel 33 5 57571742 E-mail Received 23 March 2005 revised 24 May 2005 accepted 3 June 2005 doi In our attempt to obtain further information on the replication mechanism of the hepatitis C virus HCV we have studied the role of sequences at the 3 -end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase RdRp . In this report we investigated the template and binding properties of mutated and deleted RNA fragments of the 3 -end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3 -SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3 -end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary deletions of sequences in the 5 -end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3 -end interact with HCV RdRp during in vitro RNA synthesis in particular the region located between nucleotides 219 and 239. Hepatitis C virus HCV is the major causative agent of non-A non-B hepatitis 1 . .