The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3 0 end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3 0 UTR of the (1) strand RNA. In contrast the 341 nucleotides at the 3 0 end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro.At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3 0 end of the minus-strand RNA: (a) the presence of a C residue as the 3 0 terminal nucleotide; (b) one or two G residues at positions 12 and 13; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3 0 end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.Keywords: HCV; RdRp; viral RNA; replication.Hepatitis C virus (HCV) is the major causative agent of transfusion-associated and sporadic non-A, non-B hepatitis [1]. More than 70% of HCV-infected patients develop chronic infection, often causing liver diseases such as chronic hepatitis, cirrhosis and hepatocellular carcinomas [2,3]. To date, the most effective treatment is a combination of interferon-a and the nucleoside analog ribavirin. However, only 40% of treated patients display a sustained biochemical response and inhibition of viral replication. Therefore, a more effective antiviral therapy is urgently required. HCV is a member of the family Flaviviridae. It is an enveloped virus with a single-stranded positive-sense RNA genome that contains a single long ORF translated as a polyprotein of about 3010 amino acids [4]. The ORF is flanked by two untranslated regions (UTR). The 341-nucleotide 5 0 UTR, together with the first nucleotides coding for the capsid, form an internal ribosome entry site (IRES) which is important for translation of the ORF. The 3 0 UTR is composed of a short variable region, a polyuridine tract of variable length and a 98-nucleotide sequence (3 0 X) which is highly conserved among various isolates [5]. It has been shown that this region is necessary for viral infectivity [6] but its exact role in viral replication is unknown.Studies of HCV replication have been hampered by the lack of efficient culture systems and by the fact that the only animal model is the chimpanzee [7,8]. More recently, a system allowing replication of a subgenomic fragment of hepatitis C virus RNA in a hepatoma cell line has been described [9]. Thus, most of the studies on the structures and functions of viral prote...

Initiation of translation driven by internal ribosome entry site (IRES) elements depends upon the structural organization of this mRNA region. Besides translation initiation factors (eIFs), auxiliary proteins can also affect IRES activity. With the aim to identify proteins interacting with two unrelated IRESs present in the genome of foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) we have used a proteomic approach. This procedure allowed the identification of 21 RNA-binding proteins interacting with discrete regions of the FMDV IRES, domains 3 and 5, and 16 interacting with domain III of the HCV IRES. In support of the binding specificity, the factors interacting with domain 3 differed from those interacting with domain 5, and included three poly(rC)-binding protein (PCBP) members, besides proliferation-associated 2G4 (PA2G4) and deleted-azoospermia 1 (DAZ1) protein. Around 71% of the identified factors associated with the FMDV IRES differ from those interacting with the HCV IRES. The group of proteins interacting with the FMDV or the HCV IRES includes eIF4B and 5 subunits of eIF3, respectively, known to interact with each of these RNAs, validating the results of this approach. According to the function of the identified proteins, 55% are involved in translation control, whereas 35% play a role in different aspects of RNA lifespan. Compilation of factors preferentially associated with FMDV or HCV IRES provides a basis for examining the strategies used by IRESs to recruit the translation machinery.

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.