Properties of hepatitis C virus minigenome containing mutated 5'UTR region and luciferase transgene.
Sequences at the 3'UTR of Hepatitis C virus (HCV) negative-strand (-)RNA play an important role in the initiation of positive-strand (+)RNA synthesis. However, the underlying mechanism in cellular context is still unclear. In this report, we designed several cDNA-based HCV-like minigenomes containing different mutations at the 5'UTR of (+)RNA. These (+)RNAs transcribed from the minigenomes in vitro were transfected into HCV replicon cells for producing (-)RNAs with deletions of different stem loops (SL) at the 3'-end. The results showed that expression of the antisense transgene from minigenome increased, when the minigenome containing deletion of SL-C1+D1+E1 at the 3'-end of (-)RNA was transfected into the HCV replicon cells compared to that of the full minigenome. The expression of the transgene from minigenome decreased using other mutant minigenomes containing deletions SL-A1, SL-A1+B1, and SL-A1+B1+C1 at the 3'-end of (-)RNA. Finally, the transgene from SL-C1+D1+E1 of (-)RNA using CMV promoter-driven minigenome was expressed at higher level than full minigenome in HCV replicon cell lines. These results indicated that the region of (-)RNA interacting with HCV replicase may locate in the SL-C1+D1+E1 region of (-)RNA.


BACKGROUND
Hepatitis C virus; minigenome; RNA dependent RNA polymerase; replication.
Yang
J
J
Department of Microbiology, the Fourth Military Medical University of PLA, 17 Changlexi Rd., Xi'an, Shaanxi 710032, PR, China.
Lei
Y F
YF
An
Q X
QX
Yin
W
W
Lu
X
X
Ye
L
L
Wei
S H
SH
Jia
Z
Z
Xu
Z K
ZK
D020121:5' Untranslated Regions; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection
D020121:5' Untranslated Regions; D017931:DNA Primers; D004279:DNA, Viral; D016372:RNA, Antisense; D012367:RNA, Viral; D011994:Recombinant Proteins; D008156:Luciferases