De Novo RNA Synthesis Catalyzed by HCV RNA-Dependent RNA Polymerase

Sun, Xin-Lai; Johnson, Robert B.; Hockman, Michelle A.; Wang, Q M

doi:10.1006/bbrc.2000.2120

Cited by 65 publications

(57 citation statements)

References 21 publications

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“…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.…”

Section: Discussionsupporting

confidence: 91%

“…Interestingly, we did not find that NS5B could copy a small homopolymeric oligonucleotide (U)20 by initiating RNA synthesis from the 3'-end of the template, suggesting that RNA synthesis from the 3'-UTR of HCV genome is not likely to initiate from the U-rich tract upstream of X-RNA in 3'-UTR. This result argues against a recent report showing that HCV RNA polymerase can start RNA synthesis internally from a pyrimidine base in poly(U) and the 3'-UTR of the HCV genome (Sun et al, 2000;Pellerin et al, 2002).…”

Section: Discussioncontrasting

confidence: 85%

“…Previously, insect cell-expressed full-length NS5B proteins have been shown to initiate RNA synthesis by snap-back intramolecular priming (Behrens et al, 1996;Lohmann et al, 1997) or by internal initiation in the poly(U) and 3'-UTR of the HCV genome (Sun et al, 2000;Pellerin et al, 2002), whereas the full-length HCV RNA polymerase expressed in E. coli was shown to start RNA synthesis de novo from internal single strand region in X-RNA loop I and from the 3'-end of (-) 3'-UTR of HCV genome (Blight and Rice, 1997;Oh et al, 1999). To clarify this discrepancy, RdRp reactions was performed using X-RNA and the (-) 3'-UTR of HCV.…”

Section: Rna Synthesis Initiation From X-rna and M Inus-strand 3'-utrmentioning

confidence: 99%

“…The NS5B with TNTase activity generated dimer-size RNA product using the full-length 9.6-kb HCV RNA template, which is probably the RNA product synthesized by TNTase-mediated priming and/or copy-back RNA synthesis mechanism from the X-RNA. Furthermore, two recent studies have demonstrated that E. coli-and insect cell-expressed full-length HCV NS5B proteins utilize a pyrimidine base in poly (U) and the 3'-UTR of the HCV genome for RNA synthesis initiation (Sun et al, 2000;Pellerin et al, 2002). One reason for the discrepancy with respect to RNA synthesis initiation using natural HCV cis-acting element at the 3'-UTR might be due to intrinsic TNTase activity of HCV NS5B or host origin TNTase co-purified with HCV NS5B.…”

Section: Introductionmentioning

confidence: 99%

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

Choi

Kim

Oh³

2003

Exp Mol Med

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A bstractThe hepatitis C virus (HCV) RNA-dependent RNA polym erase, NS5B protein, is the key viral enzym e responsible for replication of the HCV viral RNA genom e. Although several full-length and truncated form s of the HCV NS5B proteins have been expressed previously in insect cells, contam ination of host term inal transferase (TNTase) has ham pered analysis of the RNA synthesis initiation m echanism using natural HCV RNA tem plates. W e have expressed the HCV NS5B protein in insect cells using a recom binant baculovirus and purified it to near hom ogeneity w ithout contam inated TNTase. The highly purified recom binant HCV NS5B w as capable of copying 9.6-kb full-length HCV RNA tem plate, and m ini-HCV RNA carrying both 5'-and 3'-untranslated regions (UTRs) of the HCV genom e. In the absence of a prim er, and other cellular and viral factors, the NS5B could elongate over HCV RNA tem plates, but the synthesized products were prim arily in the double stranded form , indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA tem plates representing the 3'-end region of HCV m inus-strand RNA and the X-RNA at the 3'-end of HCV RNA genom e w as also initiated de novo. N o form ation of dim er-size self-prim ed RNA products resulting from extension of the 3'-end hydroxyl group w as observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polym erase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genom e.

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Section: Discussionsupporting

confidence: 91%

Section: Discussioncontrasting

confidence: 85%

Section: Rna Synthesis Initiation From X-rna and M Inus-strand 3'-utrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Choi

Kim

Oh³

2003

Exp Mol Med

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“…Like poliovirus (15), the HCV RdRp is capable of initiating viral RNA synthesis in vitro by a primer-dependent mechanism (9,10,16). However, Flaviviridae RdRps have also been shown to initiate RNA synthesis by a de novo mechanism (12,13,(17)(18)(19). De novo initiation of virus replication is the likely preferred mechanism for HCV in infected cells (20,21).…”

mentioning

confidence: 99%

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

Johnston

Gutshall

et al. 2003

Journal of Biological Chemistry

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In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of ⌬21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNAbound form of ⌬21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.

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Kinetic Analysis of C-Terminally Truncated RNA-Dependent RNA Polymerase of Hepatitis C Virus

Kashiwagi

Hara

Kohara

et al. 2002

Biochemical and Biophysical Research Communications

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De Novo RNA Synthesis Catalyzed by HCV RNA-Dependent RNA Polymerase

Cited by 65 publications

References 21 publications

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

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

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

Kinetic Analysis of C-Terminally Truncated RNA-Dependent RNA Polymerase of Hepatitis C Virus

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