Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells.
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.
Choi
Han-Byul
HB
Department of Biotechnology, Yonsei University, 134 Sinchon-dong, Seodaemun-gu Seoul 120-749, Korea.
Kim
Yeon-Gu
YG
Oh
Jong-Won
JW
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D016678:Genome; D016679:Genome, Viral; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013698:Templates, Genetic; D014542:Uridine Monophosphate / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D012313:RNA; D012367:RNA, Viral; D011994:Recombinant Proteins; D014542:Uridine Monophosphate; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase