Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.
BACKGROUND
Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine.


METHODS
Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-alpha (IFNalpha) was evaluated.


RESULTS
The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNalpha enhanced the antiviral effect of IFNalpha and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNalpha but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine.


CONCLUSIONS
The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.
Mizui
Tomokazu
T
Department of Gastroenterology, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. don-chip@aqua.email.ne.jp
Yamashina
Shunhei
S
Tanida
Isei
I
Takei
Yoshiyuki
Y
Ueno
Takashi
T
Sakamoto
Naoya
N
Ikejima
Kenichi
K
Kitamura
Tsuneo
T
Enomoto
Nobuyuki
N
Sakai
Tatsuo
T
Kominami
Eiki
E
Watanabe
Sumio
S
D000998:Antiviral Agents / Q000008:administration & dosage / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D016174:Hepacivirus / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000008:administration & dosage; D034741:RNA, Small Interfering / Q000008:administration & dosage; D014162:Transfection; D014764:Viral Proteins / Q000187:drug effects / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D016898:Interferon-alpha; D034741:RNA, Small Interfering; D014764:Viral Proteins; D002738:Chloroquine