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...

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "␤-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the ␤-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA. Hepatitis C virus (HCV)7 is a member of the Flaviviridae family that induces severe liver disease in humans (1). The viral genome is a single-stranded RNA of positive polarity containing a single open reading frame (ORF) flanked by two untranslated regions (UTRs), the 5Ј-UTR and 3Ј-UTR. The single ORF is translated into a large (ϳ3000 residues) polyprotein that is processed into some 10 mature proteins. Thus, the RNA-dependent RNA polymerase (RdRp) NS5b is cleaved from the C terminus of the polyprotein. In vivo the 591-residue NS5b is the central player in the synthesis of new genomic RNAs, in association with other viral and cellular proteins. This viral replication complex is associated with membranes (2) with the highly hydrophobic C-terminal 21 residues of NS5b forming a transmembrane helix (3). In vitro, NS5b has been shown to be capable of template-directed RNA synthesis on its own, requiring only divalent metals (magnesium or manganese) as cofactors. Indeed, NS5b can catalyze both de novo synthesis from a singlestranded template (4) and primer extension from the subsequent RNA duplex or from a preannealed template/primer duplex. The NS5b C-terminal transmembrane helix is dispensable for these activities, and C-terminal deletions of 21 residues (NS5b_⌬21) or more (NS5b_⌬47 to NS5b_⌬60) have been used in most activity and all crystallographic studies. The latter (5-7) has shown that the catalytic core of NS5b comprises residues 1-530 (Fig. 1E). They have also brought a puzzle to light; the 40-residue stretch (termed "linker" throughout this manuscript) between the catalytic core (fingers, palm, and thumb, Fig. 1) and the C-terminal membrane anchor occludes the catalytic cleft (5) in the crystal structures in which it is present (i.e. _⌬21 forms). The only reported exception is the case of the consensus subty...

The hepatitis C virus (HCV) 5' untranslated region (UTR) has been extensively studied with regard to its internal ribosomal entry site (IRES) activity. In this work we present results suggesting the existence of a strong promoter activity carried by the DNA sequence corresponding to the HCV 5' UTR. This activity was not detected when the HCV 5' UTR sequence was replaced by HCV 3' UTR or poliovirus 5' UTR sequences. These results were further confirmed by using bicistronic constructions. We demonstrated the presence of an mRNA initiated in this 5' UTR sequence and located the initiation site by the 5' RACE method at nucleotide 67. Furthermore, northern experiments and flow cytometry analysis showed the unambiguous activity of such a promoter sequence in stably transfected cells. Our results strongly suggest that the data obtained using bicistronic DNA constructs carrying the HCV 5' UTR should be analyzed not only at the translational but also at the transcriptional level.

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.Hepatitis C virus (HCV) infection causes serious liver diseases, such as chronic hepatitis, which can evolve into cirrhosis and hepatocellular carcinoma (20,39). The most effective therapy, a combination of pegylated interferon and ribavirin, is efficient in only 50% of patients (32). Therefore, new treatments based on specific and well-tolerated compounds need to be developed.HCV RNA replication is catalyzed by the viral polymerase NS5B. This RNA-dependent RNA polymerase (RdRp) synthesizes a negative-strand RNA that serves as a template for the synthesis of new positive RNA strands. Viral RNA synthesis can be divided into two major steps: (i) initiation, which corresponds to the formation of a 2-or 3-nucleotide (nt) product and which occurs by a de novo mechanism in vitro as well as in the cellular replicon system that mimics some steps of the in vivo viral cycle (9, 31, 49), and (ii) elongation, which yields a full copy of the template. The transition between these two steps, in which 3-nt to 5-nt products are synthesized, may involve conformational changes of the viral polymerase that are not yet understood (14). The structure of the enzyme, as determined by X-ray crystallography, revealed, like for many other RNA and DNA polymerases, a right-handed-like structure with finger, palm, and thumb domains (1,7,28). It has been shown that the NS5B protein may adopt a closed and active conformation. The contact between the two loops extending from the fingers and the thumb links these two domains and closes the back of the enzyme to form a tunnel that constitutes the entry site for ribonucleotides. This suggests that a concerted movement of the thumb and fingertips occurs during the polymerization steps. The...