Tài liệu Báo cáo khoa học: Template requirements and binding of hepatitis C virus NS5B polymerase

Template requirements and binding of hepatitis C virus

NS5B polymerase during in vitro RNA synthesis from the

3¢-end of virus minus-strand RNA

The´re` se Astier-Gin, Pantxika Bellecave, Simon Litvak and Michel Ventura

UMR-5097 CNRS, Universite´ Victor Segalen Bordeaux 2, Bordeaux, France

Hepatitis C virus (HCV) is the major causative agent

of non-A, non-B hepatitis [1]. This virus has a posit￾ive-stranded RNA genome and belongs to the Flavivir￾idae family. The RNA contains a large open reading

frame that encodes a polyprotein which is cleaved into

10 viral proteins: C, E1, E2, p7, NS2, NS3, NS4A,

NS4B, NS5A and NS5B [2]. Recently, a frame shift

product of HCV core encoding sequence, the F pro￾tein, was described [3,4]. This protein has no known

functions. The large open reading frame is flanked by

two untranslated regions (UTR). The 341-nucleotide

(nt) 5¢UTR in association with the first nucleotides of

the core protein contains an internal ribosome entry

site (IRES) that directs cap-independent translation of

the viral RNA [5,6]. The 3¢UTR is composed of a

short variable region, a polypyrimidine tract (poly

U-UC) of variable length and a highly conserved

98-nucleotide segment (3¢X). The two latter domains

are essential for viral infectivity in vivo [7] and RNA

replication of HCV in the HCV replicon system [8,9].

HCV RNA replication occurs in two steps. In the

first step the viral replicase synthesizes a minus-strand

RNA that serves as a template for the synthesis of

new plus-strand RNA molecules. Initiation of RNA

synthesis at the 3¢-end of the plus- and minus-strand

RNA most probably involves interactions between

the protein components of the replication complex,

in particular with the viral polymerase (NS5B), and

structures and ⁄ or sequences of the viral RNA

templates. The secondary structure of the 3¢-end of the

Keywords

HCV; minus strand RNA; RdRp

Correspondence:

The´re` se Astier-Gin, CNRS UMR5097,

Universite´ Victor Se´galen Bordeaux 2, 146,

rue Le´o Saignat, 33076 Bordeaux cedex,

France

Fax: +33 5 57571766

Tel: +33 5 57571742

E-mail: [email protected]

(Received 23 March 2005, revised 24 May

2005, accepted 3 June 2005)

doi:10.1111/j.1742-4658.2005.04804.x

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 sug￾gest that several domains of the 341 nucleotide region of the minus-strand

3¢-end interact with HCV RdRp during in vitro RNA synthesis, in parti￾cular the region located between nucleotides 219 and 239.

Abbreviations

HCV, hepatitis C virus; IRES, internal ribosome entry site; nt, nucleotide; RdRp, RNA-dependent RNA polymerase; TCA, trichloroacetic acid;

UTR, untranslated region.

3872 FEBS Journal 272 (2005) 3872–3886 ª 2005 FEBS