Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro

Hey, T D; Richards, Oliver C.; Ehrenfeld, Ellie

doi:10.1128/jvi.58.3.790-796.1986

Cited by 51 publications

(37 citation statements)

References 37 publications

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“…Several laboratories investigating viral RdRps in vitro obtained dimer-sized RNA products. In the case of the poliovirus RdRp 3DPol protein, this phenomenon has been described and discussed by a number of different authors (Tuschall et al, 1982;Young et al, 1985;Hey et al, 1986Hey et al, , 1987Lubinski et al, 1986;Plotch et al, 1989;Neufeld et al, 1991;Cho et al, 1993). Interestingly, in the case of polioviruses and EMCVs, dimer-sized genomic RNA molecules have also been detected in vivo (Senkevich et al, 1980;Young et al, 1985), which fuels speculations that these molecules may represent intermediates in the RNA replication process.…”

Section: Discussionmentioning

confidence: 97%

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

1996

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Hepatitis C virus (HCV) is the major etiological agent of non‐A, non‐B post‐transfusion hepatitis. Its genome, a (+)‐stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)‐strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (‐) RNA strand, which serves, in turn, as a template for the production of progeny (+)‐strand RNA molecules. An RNA‐dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA‐dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non‐structural protein 5B (NS5B). The characterization of the HCV RNA‐dependent RNA polymerase product revealed that dimer‐sized hairpin‐like RNA molecules are generated in vitro, indicating that NS5B‐mediated RNA polymerization proceeds by priming on the template via a ‘copy‐back’ mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA‐ or DNA oligonucleotide primer‐dependent RNA synthesis on templates with a blocked 3′ end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.

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Section: Discussionmentioning

confidence: 97%

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

1996

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“…In poliovirus, which has a 3Ј homopolymeric poly(A) tail with an average length of about 60 nucleotides, an oligo(U) primer is required in vitro for initiation of RNA synthesis, generating a full-length minus strand (34,40,43). Interestingly, this primer can be substituted by a host cell factor, assumed to be a terminal uridylyl transferase (13,17), which can add several uridine residues to the 3Ј end of the genome, generating an RNA molecule which can form a 3Ј-terminal loop structure. Consequently, molecules primed by this mechanism have twice the length of the genome (17).…”

Section: Discussionmentioning

confidence: 99%

Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity

Lohmann

Körner

Herian

et al. 1997

J Virol

482

273

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The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.

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“…A total of 300 ng (4.6 pmol) of polymerase was assayed in a 50-µL reaction mixture containing 20 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 10 mmol/L MgCl 2 , 10 mmol/L NaCl, 1 mmol/L DTT, 2,000 U of RNase inhibitor (Takara Shuzo) per milliliter, 50 µg of Actinomycin D per milliliter, 10 µg of poly(A) (Yamasa-shoyu, Chiba, Japan) per milliliter, and 25 µg of oligo(U) 15 (Takara Shuzo) per milliliter. 36 As the labeling nucleotide, 2.5 µCi of [ 3 H]-labeled UTP (Daiichi Kagaku, Tokyo, Japan) was used. After a 2-hour incubation at 32°C, 8 µL of reaction solutions was spotted on a filtermat (Wallac, Turku, Finland) and dried completely in an 80°C oven, followed by washing three times with 2ϫ standard saline citrate (300 mmol/L sodium chloride and 30 mmol/L sodium citrate) and twice with 95% ethanol.…”

Section: Methodsmentioning

confidence: 99%

Expression of hepatitis C virus NS5B protein: Characterization of its RNA polymerase activity and RNA binding

Ishii¹,

Tanaka²,

Yap³

et al. 1999

Hepatology

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The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U) 15 ) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32ЊC and pH 8. Hepatitis C virus (HCV) is the major etiologic agent of both posttransfusion and sporadic non-A, non-B hepatitis throughout the world. 1-3 HCV infection often leads to persistence, resulting in chronic hepatitis, and develops into hepatocellular carcinoma. Although the incidence of infection with HCV was drastically decreased by introduction of an efficient blood screening system, 1% to 2% of the worldwide population are already infected with HCV. Furthermore, seroepidemiological studies indicated that there is a clear correlation between infection with HCV and the development of hepatocellular carcinoma. It is therefore very important to develop anti-HCV agents with different antiviral mechanisms for the treatment of liver diseases associated with HCV infection. A number of other drugs with a broad spectrum of antiviral activity have been given to patients with hepatitis C. 4 To date, however, few convincing positive results have been obtained.HCV is a positive-stranded RNA virus with the genome size of approximately 9,400 bases and contains a large open reading frame encoding a precursor polyprotein of about 3,000 amino acids. [5][6][7][8] From their deduced amino acid sequence, HCV was shown to be related to pestiviruses and distantly related to flaviviruses. [8][9][10][11] Recent studies revealed that this polyprotein was cleaved into at least 9 mature proteins by host cellular signalases and viral coded proteinases. 12,13 However, because of the lack of an efficient in vitro cell culture system to grow the virus, little is known about the mechanisms of the replication of HCV. Synthesis of both replicative intermediate minus-strand HCV RNA and progeny positive-strand HCV RNA is thought to occur via an RNA-dependent RNA polymerase (RdRp) reaction. The nonstructural 5B (NS5B) protein of 65 kd is located at the C-terminal region of the HCV polyprotein and shares its sequence with those of other proteins of animal and plant viruses and bacteriophages possessing RdRp activity. 14,15

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Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro

Cited by 51 publications

References 37 publications

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity

Expression of hepatitis C virus NS5B protein: Characterization of its RNA polymerase activity and RNA binding

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