Abstract
BackgroundHepatitis 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.MethodsWe 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.ResultsIn 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.ConclusionsHCV 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.
Highlights
Basic cell functions such as proliferation, growth, differentiation, autophagy and glucose and lipid metabolism show time related fluctuations, and when the oscillations are rhythmic with a periodicity of approximately 24 h the rhythmicity is defined circadian [1]
As viruses are highly dependent on cellular machinery for replication, it was proposed that the viral replication may be synchronized to the molecular clockwork and that in turn the circadian clock may influence viral replication [7]
With this premise in mind, we sought to analyze by qRT-PCR the time-qualified expression of a panel of clock genes (Rev-Erba, Rora, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in OR6 cells, which express full length Hepatitis C virus (HCV) replicon of genotype 1b [16]
Summary
Basic cell functions such as proliferation, growth, differentiation, autophagy and glucose and lipid metabolism show time related fluctuations, and when the oscillations are rhythmic with a periodicity of approximately 24 h the rhythmicity is defined circadian [1]. Cellular circadian rhythmicity is driven by molecular clockworks comprised of translational-transcriptional feedback loops put in place by a set of genes, called core clock genes, coding for proteins that in turn suppress gene expression in a cycle that completes itself in one day. E4BP4, a transcription factor that regulates mammalian circadian oscillatory mechanism, coordinates expression of viral genes with the cellular molecular clock and represses viral promoter sequences [7,8]. 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. The relation between HCV replication and the circadian clock is unknown
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