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

Han Byul Choi, Yeon Gu Kim, Jong Won Oh

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5′- and 3′-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3′-end region of HCV minus-strand RNA and the X-RNA at the 3′-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3′-end hydroxyl group was 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 polymerase initiates RNA synthesis from the selected region in the 3′-UTR of HCV genome.

Original languageEnglish
Pages (from-to)475-485
Number of pages11
JournalExperimental and Molecular Medicine
Volume35
Issue number6
DOIs
Publication statusPublished - 2003 Dec 31

Fingerprint

RNA Replicase
Viruses
Hepacivirus
Insects
RNA
Genes
3' Untranslated Regions
Genome
Transferases
Insect Proteins
Copying
Proteins
Baculoviridae
Viral Genome
5' Untranslated Regions
Viral RNA
DNA-Directed RNA Polymerases
Hydroxyl Radical
Dimers

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry

Cite this

@article{fd3647a6391641358684acc67936655d,
title = "Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells",
abstract = "The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5′- and 3′-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3′-end region of HCV minus-strand RNA and the X-RNA at the 3′-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3′-end hydroxyl group was 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 polymerase initiates RNA synthesis from the selected region in the 3′-UTR of HCV genome.",
author = "Choi, {Han Byul} and Kim, {Yeon Gu} and Oh, {Jong Won}",
year = "2003",
month = "12",
day = "31",
doi = "10.1038/emm.2003.62",
language = "English",
volume = "35",
pages = "475--485",
journal = "Experimental and Molecular Medicine",
issn = "1226-3613",
publisher = "Korean Society of Med. Biochemistry and Mol. Biology",
number = "6",

}

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells. / Choi, Han Byul; Kim, Yeon Gu; Oh, Jong Won.

In: Experimental and Molecular Medicine, Vol. 35, No. 6, 31.12.2003, p. 475-485.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

AU - Choi, Han Byul

AU - Kim, Yeon Gu

AU - Oh, Jong Won

PY - 2003/12/31

Y1 - 2003/12/31

N2 - The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5′- and 3′-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3′-end region of HCV minus-strand RNA and the X-RNA at the 3′-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3′-end hydroxyl group was 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 polymerase initiates RNA synthesis from the selected region in the 3′-UTR of HCV genome.

AB - The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5′- and 3′-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3′-end region of HCV minus-strand RNA and the X-RNA at the 3′-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3′-end hydroxyl group was 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 polymerase initiates RNA synthesis from the selected region in the 3′-UTR of HCV genome.

UR - http://www.scopus.com/inward/record.url?scp=1642542018&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1642542018&partnerID=8YFLogxK

U2 - 10.1038/emm.2003.62

DO - 10.1038/emm.2003.62

M3 - Article

C2 - 14749524

AN - SCOPUS:1642542018

VL - 35

SP - 475

EP - 485

JO - Experimental and Molecular Medicine

JF - Experimental and Molecular Medicine

SN - 1226-3613

IS - 6

ER -