cDNA microarray analysis to compare HCV subgenomic replicon cells with their cured cells.
The hepatitis C virus (HCV) replicon system carrying autonomously replicating HCV subgenomic RNA in human hepatocyte cells is a potent tool for basic studies of HCV, such as viral replication and drug development. Recently, we developed two HCV subgenomic replicons (50-1 and 1B-2R1) derived from two HCV strains, 1B-1 and 1B-2, respectively. Since the expression of HCV proteins is thought to affect the host cells' gene expression profiles, we attempted to identify target genes of HCV proteins using microarray analysis (9970 genes) by comparing 50-1 and 1B-2R1 replicon cells with their "cured cells", from which the replicons had been eliminated by prolonged treatment with interferon-alpha. The results showed that HCV replicons could have a variety of expression profiles in human hepatocytes. The results also showed that 2 and 6 genes were commonly up-regulated (more than 2.0-fold) and down-regulated (less than 0.50-fold), respectively, in both 50-1 and 1B-2R1 replicon cells compared with their cured cells. The differential expression profiles of genes selected by the microarray analysis were confirmed with standard RT-PCR and real-time LightCycler PCR. It was noteworthy that the commonly down-regulated genes contained large multifunctional proteases 2 and 7, which are known as catalytic subunits of immunoproteasome, and serine proteinase inhibitor clade C. Our microarray analysis demonstrated that HCV subgenomic replicons can change the gene expression profiles of host cells, and it allowed us to compile the first list of genes that the replicons transcriptionally regulate.
Abe
Ken-ichi
K
Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Ikeda
Masanori
M
Dansako
Hiromichi
H
Naka
Kazuhito
K
Shimotohno
Kunitada
K
Kato
Nobuyuki
N
D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics*; D020869:Gene Expression Profiling; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000235:genetics
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral