転写因子JunBはC型肝炎ウイルスの複製を抑制する

概要

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PDF issue: 2024-05-02

Transcription factor JunB suppresses hepatitis
C virus replication

ADI ARIFFIANTO
(Degree)
博士（医学）

(Date of Degree)
2023-09-25

(Resource Type)
doctoral thesis

(Report Number)
甲第8723号

(URL)
https://hdl.handle.net/20.500.14094/0100485907
※ 当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

（課程博士関係）

学 位 論 文 の内 容 要 旨

Transcription factor JunB suppresses hepatitis C virus replication

転写因子 JunB は C 型肝炎ウイルスの複製を抑制する

神戸大学大学院医学研究科医科学専攻
微生物感染症学講座 感染制御学
（指導教員：勝二 郁夫 教授）

ADI ARIFFIANTO

SUMMARY
Background:
According to the World Health Organization, an estimated 58 million people
worldwide are chronically infected with hepatitis C virus (HCV), and 1.5 million new infections
occur each year, highlighting that HCV remains a significant public health concern. HCV is a
leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Despite the fact
that direct-acting antivirals (DAAs) therapy has a high sustained virological response rate of
over 95% in patients with various HCV genotypes, the emergence of DAAs resistance and
limited access to DAAs therapy in developing countries continue to pose challenges to global
efforts to eliminate HCV. HCV is a positive-sense single-stranded RNA virus that belongs to
the Hepacivirus genus of the Flaviviridae family. The HCV genome consists of 9.6-kb RNA
encoding a single polyprotein of about 3,010 amino acids, which is processed by viral proteases
and cellular signalases to produce three structural proteins (Core, E1, and E2) and seven
nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).
We previously reported that HCV infection induces reactive oxygen species (ROS)
production and activates the c-Jun N-terminal kinase (JNK) signaling pathway, leading to the
enhancement of hepatic gluconeogenesis and the induction of apoptosis. Recently, we
demonstrated that the HCV-mediated ROS/JNK signaling pathway promotes the release of
HCV particles via activation of the E3 ubiquitin ligase Itch. The activated JNK pathway
regulates various physiological processes, such as cell death, differentiation, proliferation, and
carcinogenesis through phosphorylation of numerous targets, including JunB, a member of the
activator protein-1 (AP-1) transcription factor family. Transcription factor JunB has been
implicated in controlling proliferation, apoptosis, and malignant transformation by regulating
transcription of target genes, such as cyclin-dependent kinase inhibitor p16 (INK4a) and matrix
metalloproteinase 2. However, the roles of JunB in the HCV life cycle and HCV-associated
pathogenesis are largely unknown.

In this study, we demonstrate that the HCV-induced ROS/JNK signaling pathway activates
the transcription factor JunB. We demonstrate evidence suggesting that JunB plays roles in
inhibiting HCV replication and upregulation of hepcidin. Thus, we propose that the
ROS/JNK/JunB signaling pathway negatively regulates HCV replication and also contributes
to iron metabolism disorder via the activation of hepcidin transcription.
Methods:
1. To determine whether HCV infection induces phosphorylation and activation of JunB. We
infected Huh-7.5 cells with HCV genotype 2a J6/JFH1 and detect the phosphorylation levels
of JunB by immunoblotting. We treated the cells with daclatasvir, an HCV NS5A inhibitor,
to eliminate HCV and detect the phosphorylation of JunB to further verify the involvement
of HCV in the phosphorylation of JunB.
2. To determine whether ROS/JNK signaling pathway induces JunB activation, we treated HCVinfected cells with either the antioxidant NAC 5 mM for 8 hours or the specific JNK inhibitor
SP600125 30 µM for 30 hours. We performed immunoblot analysis to determine the
phosphorylation status of JunB in HCV-infected cells.
3. To clarify the role of JunB in HCV replication. We transfected JunB siRNA or JunB
expression plasmid to knockdown endogenous JunB or overexpressed JunB in HCV J6/JFH1
-infected Huh-7.5 cells and Huh-7 cells stably harboring an HCV-1b FGR derived from Con1
(RCYM1). We examined the amount of intracellular viral protein by western blotting, HCV
RNA by Real-time RT-QPCR, and HCV infectivity titers as well as extracellular HCV
infectivity titers by immunostaining.
4. To elucidate a role of JunB in HCV-mediated iron metabolism disorder, we transfected JunB
siRNA or JunB expression plasmid together with pGL4.10-Hepcidin promoter and pRLCMV-Renilla in both HCV- or mock-infected cells. Luciferase reporter assay was performed
using a dual-luciferase reporter assay system (Promega) to measure the hepcidin promoter
activity.
5. To determine the effect of HCV infection on hepcidin mRNA levels, we infected Huh-7.5
cells with HCV and performed real-time RT-PCR assays.
Results:
1. Immunoblot analysis showed that the amount of activated JunB phosphorylated at

Thr102/Thr104 and the total expression levels of JunB markedly increased in HCV J6/JFH1infected Huh-7.5 cells compared to those in the mock-infected control cells. Moreover,
treatment of the cells with daclatasvir reduced the HCV-induced phosphorylation of JunB at
Thr102/Thr104. These results indicate that HCV infection promotes the phosphorylation of
JunB.
2. The HCV-induced JunB phosphorylation at Thr102/Thr104 was markedly reduced after
treatment with SP600125 or NAC. In parallel, treatment with SP600125 or NAC clearly
decreased the phosphorylation of c-Jun and the total c-Jun protein levels, a key substrate for
JNK. These results suggest that HCV infection promotes phosphorylation and activation of
JunB via the ROS/JNK signaling pathway.
3. Knockdown of JunB increased the level of intracellular HCV NS3 protein, the amount of
intracellular HCV RNA, intracellular HCV infectivity titers, and extracellular HCV
infectivity titers. Conversely, overexpression of JunB reduced the level of intracellular HCV
NS3 protein, the amount of intracellular HCV RNA, intracellular HCV infectivity titers, and
extracellular HCV infectivity titers. These results suggest that JunB suppresses HCV
replication in both HCV genotypes 2a and 1b.
4. Luciferase promoter assays demonstrated a significant enhancement of hepcidin promoter
activity in HCV-infected Huh-7.5 cells compared to mock-infected control cells. Notably,
knockdown of JunB significantly disrupted the HCV-induced enhancement of hepcidin
promoter activity. Conversely, overexpression of JunB markedly augmented the HCVinduced enhancement of hepcidin promoter activity compared to control cells. These results
suggest that JunB is involved in HCV-induced enhancement of hepcidin promoter activity.
5. Real-time RT-PCR assays showed that HCV infection increased hepcidin mRNA levels.
Knockdown of JunB reduces the HCV-induced enhancement of hepcidin mRNA levels. In
addition, overexpression of JunB increases the HCV-induced enhancement of hepcidin
mRNA levels. These results suggest that JunB is involved in HCV-induced enhancement of
hepcidin mRNA levels in HCV-infected Huh-7.5 cells.
Discussion:
In this study, we obtained results suggesting that HCV infection promotes
phosphorylation of the transcription factor JunB via the ROS/JNK signaling pathway.

Importantly, the present study suggests that JunB plays a role in inhibiting HCV replication
in both HCV genotypes 2a and 1b. To explore the role of JunB in HCV-mediated iron
metabolism disorder, we investigated the effect of JunB on hepcidin transcription in HCVinfected cells. The luciferase promoter assay and real-time RT-PCR analyses revealed that
HCV infection significantly enhanced hepcidin promoter activity and hepcidin mRNA levels.
Furthermore, our results suggest that JunB participates in HCV-induced hepcidin promoter
activity and hepcidin mRNA levels, indicating that JunB is involved in facilitating hepcidin
transcription upon HCV infection. Collectively, our results suggest that the ROS/JNK/JunB
signaling pathway plays roles in inhibiting HCV replication and contributing to HCVmediated iron metabolism disorder.
It was reported that JNK facilitates IL-1 beta-induced hepcidin transcription via the
cAMP response element site B on the hepcidin promoter in hepatocytes without HCV
infection. Our current findings demonstrated the involvement of JunB in HCV-mediated iron
metabolism disorder by enhancing hepcidin transcription. To the best of our knowledge, this
is the first study to elucidate the role of JunB in the HCV-mediated iron metabolism disorder.
Clinical studies have suggested a close link between HCV and iron metabolism
disorder, with iron accumulation in the liver tissue of patients with chronic hepatitis C. It is
widely recognized that HCV disrupts iron metabolism by regulating hepcidin levels.
Hepcidin regulates iron metabolism by interacting with ferroportin, the sole iron exporter,
and promoting degradation of ferroportin, thereby controlling the primary influx of iron into
plasma. Clinical studies have reported higher levels of hepcidin in the serum of both acute
and chronic HCV patients, which is consistent with our present data demonstrating that HCV
infection significantly increases hepcidin promoter activity and hepcidin mRNA levels.
Our results suggest that JunB inhibits HCV replication, although the underlying
mechanism remains to be determined. A previous study has demonstrated that hepcidin
inhibits HCV replication by activating the STAT3 pathway. Furthermore, increased hepcidin
expression can lead to elevated hepatic iron storage, and excess iron can inhibit the enzymatic
activity of the HCV NS5B RNA polymerase, thereby blocking HCV replication. Thus, we
speculate that the JunB-induced enhancement of hepcidin levels plays a negative role in HCV
replication by increasing the amount of iron in HCV-infected cells. We speculate that JunB
may function as a cellular defense mechanism against HCV infection through the activation

of JunB target genes, such as hepcidin. Further investigation is needed to explore the
relationship between elevated hepcidin mRNA levels and HCV replication.
In this study, we demonstrate that the HCV-induced ROS/JNK signaling pathway
activates the transcription factor JunB, which plays roles in inhibiting HCV replication and
contributing to HCV-mediated enhancement of hepcidin transcription. Thus, we propose that
the ROS/JNK/JunB signaling pathway negatively regulates HCV replication and also
contributes to HCV-mediated iron metabolism disorder.

神戸大学大学院医学（
系）
研究科 （博士課程）
言命コと 苓皆
・ 予距 クつ 糸吉 長艮 ク＞ 彦匡 旨音

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ADIARIFFIANTO

転写因子 J
unBは C型肝炎ウ イルスの複製を抑制する
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