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...
(1987) BiolTechnology 5,486-4891 has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALyS was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALyS against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVa', is observed when viral RNA or activated DNA are used as templates.Human immunodeficiency virus (HIV) is widely recognized as the etiological agent of the acquired immunodeficiency syndrome [l, 21. This retrovirus encodes an RNAdependent DNA polymerase or reverse transcriptase which copies its genomic RNA into complementary DNA using a lysine-specific tRNA as primer (tRNAiY5) [3]. Retroviral-directed DNA synthesis is initiated from the 3'hydroxyl of the terminal adenosine of the primer tRNA.Experimental evidence has accumulated over the last ten years, mainly in the avian retroviral system, indicating that avian reverse transcriptase forms a stable and specific complex with primer tRNATrp [4-81. In our laboratory we have studied the topography of the complex between avian myeloblastosis virus (AMV) reverse transcriptase and beef tRNATrp, as well as the role of the AMV reverse transcriptase in the annealing of primer tRNA to the AMV genome after partial unwinding of primer tRNA [9, lo]. Moreover, we showed that AMV reverse transcriptase was able to deacylate tryptophanyl-tRNA, thus increasing the number of free 3'-OH-tRNA termini able to initiate cDNA synthesis [ l l ] (for a Correspondence zo L.
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