HCV RNA‐dependent RNA polymerase replicates in vitro the 3′ terminal region of the minus‐strand viral RNA more efficiently than the 3′ terminal region of the plus RNA

Abstract

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...

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

“…This result suggests that Cterminal region of NS5B might play a role in the initiation of RNA synthesis from 3'-end of HCV genome. To date, HCV NS5B polymerase has been shown to initiate RNA synthesis by snap-back or de novo mechanisms using a natural HCV RNA template (Behrens et al, 1996;Lohmann et al, 1997;Yamashita et al, 1998;Ishii et al, 1999;Oh et al, 2000;Sun et al, 2000;Ranjith-Kumar et al, 2001;Reigadas et al, 2001;Kashiwagi et al, 2002;Kim et al, 2002;Pellerin et al, 2002). Our results shown with several HCV RNA templates using TNTase-free NS5B are in agreement with the previous result obtained with E. coli-expressed NS5B (Oh et al, 1999), indicating that HCV NS5B proteins initiate RNA synthesis in a primer-independent manner.…”

“…In addition, the (-) 3'-UTR was found to serve as a better template for NS5B, supporting the observation that more plus-strand HCV genome accumulates in the infected cells than the minus-strand HCV RNA (Lohmann et al, 1999). Similar result has also been obtained with C-terminal 21 amino acid truncated NS5B (Reigadas et al, 2001), but the NS5B produced a longer than template sized product using X-RNA as a template. This result suggests that Cterminal region of NS5B might play a role in the initiation of RNA synthesis from 3'-end of HCV genome.…”

“…Even though the C-terminal 21 hydrophobic amino acids are involved in membrane anchorage of the HCV NS5B in vivo (Ivashkina et al, 2002), HCV RNA synthesis initiation seems to be influenced in vitro by this hydrophobic C-terminal domain of HCV NS5B. E. coli-expressed C-terminal 21 amino acid truncated NS5B generated a longer-than-template sized product with X-RNA (Reigadas et al, 2001). Another TNTase-free HCV NS5B with deletion of the 21 C-terminal hydrophobic amino acids copied HCV X-RNA inefficiently by internal initiation (Kashiwagi et al, 2002).…”

“…Their secondary structures have been determined (Blight and Rice, 1997;Schuster et al, 2002). The promoter in the minus-strand HCV RNA has been shown to be stronger than the promoter in the 3'-UTR of plus-strand HCV genome (Reigadas et al, 2001).…”

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

“…The interaction of the 5Ј-TR 230nt with 3Ј-UTR 454nt containing either wild type or complementary mutant CYC motifs was necessary for activation of the DEN2 3Ј-UTR(ϩ) RNA template (40,41). On the other hand, hepatitis C virus NS5B polymerase initiates RNA synthesis from the 3Ј-TR of the (Ϫ)-strand viral RNA more efficiently than the 3Ј-TR of the (ϩ)-strand polarity (47). Therefore, we sought to examine the template efficiency of 3Ј-TR 230nt WNV RNA of (Ϫ)-strand polarity in comparison with the 3Ј-UTR 631nt RNA of (ϩ)-strand polarity.…”