Analysis of the 5' end structure of HCV subgenomic RNA replicated in a Huh7 cell line.
OBJECTIVE
Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5' end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate.


METHODS
HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5' end.


RESULTS
Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase.


CONCLUSIONS
It is suggested that the 5' end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5' end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5' end. This result indicates that the 5' end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.
Takahashi
Hitoshi
H
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan.
Yamaji
Masashi
M
Hosaka
Masahiro
M
Kishine
Hiroe
H
Hijikata
Makoto
M
Shimotohno
Kunitada
K
D000249:Adenosine Monophosphate / Q000032:analysis; D045744:Cell Line, Tumor; D016679:Genome, Viral; D006157:Guanosine Monophosphate / Q000032:analysis; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D012093:Replicon; D014779:Virus Replication
D012367:RNA, Viral; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases