Mutual Antagonism between Circadian Protein Period 2 and Hepatitis C Virus Replication in Hepatocytes

Gastroenterology Unit, IRCCS "Casa Sollievo della Sofferenza", Hospital San Giovanni Rotondo (FG), San Giovanni, Italy.
PLoS ONE (Impact Factor: 3.23). 04/2013; 8(4):e60527. DOI: 10.1371/journal.pone.0060527
Source: PubMed


Hepatitis C virus (HCV) infects approximately 3% of the world population and is the leading cause of liver disease, impacting hepatocyte metabolism, depending on virus genotype. Hepatic metabolic functions show rhythmic fluctuations with 24-h periodicity (circadian), driven by molecular clockworks ticking through translational-transcriptional feedback loops, operated by a set of genes, called clock genes, encoding circadian proteins. Disruption of biologic clocks is implicated in a variety of disorders including fatty liver disease, obesity and diabetes. The relation between HCV replication and the circadian clock is unknown.
We investigated the relationship between HCV core infection and viral replication and the expression of clock genes (Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in two cellular models, the Huh-7 cells transiently expressing the HCV core protein genotypes 1b or 3a, and the OR6 cells stably harboring the full-length hepatitis C genotype 1b replicon, and in human liver biopsies, using qRT-PCR, immunoblotting, luciferase assays and immunohistochemistry.
In Huh-7 cells expressing the HCV core protein genotype 1b, but not 3a, and in OR6 cells, transcript and protein levels of PER2 and CRY2 were downregulated. Overexpression of PER2 led to a consistent decrease in HCV RNA replicating levels and restoration of altered expression pattern of a subset of interferon stimulated genes (ISGs) in OR6 cells. Furthermore, in liver biopsies from HCV genotype 1b infected patients, PER2 was markedly localized to the nucleus, consistent with an auto-inhibitory transcriptional feedback loop.
HCV can modulate hepatic clock gene machinery, and the circadian protein PER2 counteracts viral replication. Further understanding of circadian regulation of HCV replication and rhythmic patterns of host-hosted relationship may improve the effectiveness of HCV antiviral therapy. This would extend to hepatic viral infections the current spectrum of chronotherapies, implemented to treat metabolic, immune related and neoplastic disease.

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Available from: Manlio Vinciguerra, Sep 30, 2015
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    • "BxPC-3 and PANC-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 ng/ml streptomycin (Invitrogen Life Technologies, Milan, Italy) while CFPAC-1 and MIA-PaCa-2 were maintained in Gibco Õ RPMI 1640 medium (Invitrogen Life Technologies, Milan, Italy) all maintained at 37 C in 5% CO 2 atmosphere incubator. Cell lines were serum shocked with 50% FBS (serum-rich medium) for 2 h in order to achieve cellular synchronization as previously described (Benegiamo et al., 2013; Pazienza et al., 2012). After 2 h, the medium used for serum shock was replaced either with normal medium supplemented with 10% FBS or with medium without FBS, and the cells were harvested at the indicated time points over 28 h. "
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    ABSTRACT: Pancreatic cancer (PC), the fourth leading cause of cancer-related deaths, is characterized by high aggressiveness and resistance to chemotherapy. Pancreatic carcinogenesis is kept going by derangement of essential cell processes, such as proliferation, apoptosis, metabolism and autophagy, characterized by rhythmic variations with 24-h periodicity driven by the biological clock. We assessed the expression of the circadian genes ARNLT, ARNLT2, CLOCK, PER1, PER2, PER3, CRY1, CRY2 and the starvation-activated histone/protein deacetylase SIRT1 in 34 matched tumor and non-tumor tissue specimens of PC patients, and evaluated in PC derived cell lines if the modulation of SIRT1 expression through starvation could influence the temporal pattern of expression of the circadian genes. We found a significant down-regulation of ARNLT (p = 0.015), CRY1 (p = 0.013), CRY2 (p = 0.001), PER1 (p < 0.0001), PER2 (p < 0.001), PER3 (p = 0.001) and SIRT1 (p = 0.017) in PC specimens. PER3 and CRY2 expression levels were lower in patients with jaundice at diagnosis ( < 0.05). Having adjusted for age, adjuvant therapy and tumor stage, we evidenced that patients with higher PER2 and lower SIRT1 expression levels showed lower mortality (p = 0.028). Levels and temporal patterns of expression of many circadian genes and SIRT1 significantly changed upon serum starvation in vitro, with differences among four different PC cell lines examined (BXPC3, CFPAC, MIA-PaCa-2 and PANC-1). Serum deprivation induced changes of the overall mean level of the wave and amplitude, lengthened or shortened the cycle time and phase-advanced or phase-delayed the rhythmic oscillation depending on the gene and the PC cell line examined. In conclusion, a severe deregulation of expression of SIRT1 and circadian genes was evidenced in the cancer specimens of PC patients, and starvation influenced gene expression in PC cell lines, suggesting that the altered interplay between SIRT1 and the core circadian proteins could represent a crucial player in the process of pancreatic carcinogenesis.
    Chronobiology International 03/2015; 32(4):1-16. DOI:10.3109/07420528.2014.1003351 · 3.34 Impact Factor
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    • "Pancreata from the four experimental mice groups were lysed and processed for immunoblotting analysis with specific CLOCK and BMAL1 antibodies as previously described [30], [31]. We pooled together (n = 4) and analyzed equal amounts of proteins lysates of pancreata for each group/time point, as previously reported [32]. "
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    PLoS ONE 03/2014; 9(3):e89505. DOI:10.1371/journal.pone.0089505 · 3.23 Impact Factor
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    • "Immunohistochemistry staining was performed as previously described [22], [23]. Briefly, four-µm-thick frozen sections of human liver were cut, dried overnight at room temperature, fixed in acetone and washed in PBS. "
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    PLoS ONE 09/2013; 8(9):e72928. DOI:10.1371/journal.pone.0072928 · 3.23 Impact Factor
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