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Alcohol could compromise the anti-hepatitis C virus (HCV) function of interferon-alpha (IFN-α). However, little information is available about the effect of alcohol on interferon-lambda (IFN-λ, type III IFN), a novel candidate for development of therapy for HCV infection. Huh7 cells were infected with HCV JFH-1 virus, then treated with alcohol, and/or IFN-λ1. RT-PCR and Western blot were used to detect the levels of HCV and key cellular factors. Overexpression or silencing expression was performed to verify the role of key factors in alcohol-attenuated anti-HCV function of IFN-λ1. Alcohol treatment compromised anti-HCV effect of IFN-λ1 in HCV JFH-1-infected Huh7 cells, evidenced by the significantly increased levels of HCV RNA, and HCV core protein in alcohol-/IFN-λ1-treated cells compared to cells with IFN-λ1 treatment alone. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol enhanced the expression of protein inhibitor of activated STAT (PIASy). Overexpression of PIASy compromised anti-HCV ability of IFN-λ1, whereas silencing expression of PIASy partly restored the alcohol-attenuated anti-HCV effect of IFN-λ1. More importantly, overexpression of PIASy significantly down-regulated the level of IFN-λ1-indcued phosphorylation of STAT1 (p-STAT1), an important adaptor in IFN-λ pathway, as well as reduced the expression of IFN-λ1-induced IFN-stimulated genes 56 (ISG56), and myxovirus resistance 1 (Mx1), two antiviral effectors in in IFN-λ pathway. These findings indicate that alcohol, through inducing the expression of negative regulator in IFN-λ pathway, inhibits IFN-λ-mediated anti-HCV action in human hepatic cells, which may lead to the poor efficacy of IFN-λ-based therapy against HCV infection.

Aims Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. Methods HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. Results Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B–I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. Conclusions: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

Both alcohol and hepatitis C virus (HCV) infection could induce cellular autophagy in liver cells, which is considered to be essential for productive HCV replication. However, whether alcohol-induced autophagy is involved in the pathogenesis of HCV infection is still poorly understood. Alcohol treatment could induce autophagy in Huh7 cells (a hepatoma cell line that supports HCV JFH-1 replication), evidenced by the increase of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the decrease of p62 level in alcohol-treated cells compared with control cells. Alcohol treatment also significantly increased PIASy (a member of the PIAS family) expression, which can act as a SUMO (small ubiquitin-like modifier protein) E3 ligase to regulate a broader range of cellular processes including autophagy. Overexpression or the silencing expression of PIASy in alcohol-treated Huh7 cells could increase or decrease autophagic activation caused by alcohol treatment, respectively, and thus affect HCV replication correspondingly. In the absence of alcohol, overexpression or silencing expression of PIASy increase or decrease the level of cellular autophagy, judged by the changes of LC3B-II and p62 levels in the presence or absence of chloroquine (CQ), a lysosome inhibitor. More importantly, in the presence of 3-methyladenine (3-MA), an inhibitor in the early stage of autophagy, the effects of overexpression or silencing expression of PIASy on HCV replication were largely blocked. Furthermore, PIASy could selectively drive the accumulation of SUMO1-conjugated proteins, along with upregulation of the expression of several important autophagy factors, including ATG7 and ATG5–ATG12. In conclusion, alcohol promotes HCV replication through activation of autophagy in Huh7 cells, which partly attributes to its induction of PIASy expression. PIASy-enhanced accumulation of SUMO1-conjugated proteins may contribute to its inducing effect of autophagy. Our findings provide a novel mechanism for the action of alcohol-promoting HCV replication in the context of cellular autophagy.