Search

Abstract For the development of antiviral agents to eliminate hepatitis B virus (HBV), it is essential to establish an HBV cell culture system that can easily monitor HBV infection. Here, we created a novel HBV infection monitoring system using a luminescent 11‐amino acid reporter, the high‐affinity subunit of nano‐luciferase binary technology (HiBiT). The HiBiT‐coding sequence was inserted at the N‐terminus of preS1 in a 1.2‐fold plasmid encoding a genotype C HBV genome. After transfection of HepG2 cells with this HiBiT‐containing plasmid, the supernatant was used to prepare a recombinant cell culture‐derived virus (HiBiT‐HBVcc). Primary human hepatocytes (PXB) were inoculated with HiBiT‐HBVcc. Following inoculation, intracellular and extracellular HiBiT activity and the levels of various HBV markers were determined. Reinfection of naive PXB cells with HiBiT‐HBVcc prepared from HiBiT‐HBVcc‐infected PXB cells was analyzed. When PXB cells were infected with HiBiT‐HBVcc at several titers, extracellular HiBiT activity was detected in a viral titer‐dependent manner and was correlated with intracellular HiBiT activity. Inhibitors of HBV entry or replication suppressed extracellular HiBiT activity. Viral DNA, RNA, and proteins were detectable, including covalently closed circular DNA, by Southern blot analysis. The synthesis of relaxed‐circular DNA from single‐stranded DNA in HiBiT‐HBV decreased to one third of that of wild‐type HBV, and the infectivity of HiBiT‐HBVcc decreased to one tenth of that of wild‐type HBVcc. HiBiT‐HBVcc prepared from PXB cells harboring HiBiT‐HBV was able to infect naive PXB cells. Conclusions: Recombinant HiBiT‐HBV can undergo the entire viral life cycle, thus facilitating high‐throughput screening for HBV infection in vitro using supernatants. This system will be a powerful tool for developing antiviral agents.

The circadian rhythm of the liver plays an important role in maintaining its metabolic homeostasis. We performed comprehensive expression analysis of microRNAs (miRNAs) using TaqMan polymerase chain reaction of liver biopsy tissues to identify the miRNAs that are significantly up‐regulated in advanced chronic hepatitis C (CHC). We found miR‐10a regulated various liver metabolism genes and was markedly up‐regulated by hepatitis C virus infection and poor nutritional conditions. The expression of miR‐10a was rhythmic and down‐regulated the expression of the circadian rhythm gene brain and muscle aryl hydrocarbon receptor nuclear translocator‐like 1 (Bmal1) by directly suppressing the expression of RA receptor‐related orphan receptor alpha (RORA). Overexpression of miR‐10a in hepatocytes blunted circadian rhythm of Bmal1 and inhibited the expression of lipid synthesis genes (sterol regulatory element binding protein [SREBP]1, fatty acid synthase [FASN], and SREBP2), gluconeogenesis (peroxisome proliferator‐activated receptor gamma coactivator 1 alpha [PGC1α]), protein synthesis (mammalian target of rapamycin [mTOR] and ribosomal protein S6 kinase [S6K]) and bile acid synthesis (liver receptor homolog 1 [LRH1]). The expression of Bmal1 was significantly correlated with the expression of mitochondrial biogenesis‐related genes and reduced Bmal1 was associated with increased serum alanine aminotransferase levels and progression of liver fibrosis in CHC. Thus, impaired circadian rhythm expression of Bmal1 by miR‐10a disturbs metabolic adaptations, leading to liver damage, and is closely associated with the exacerbation of abnormal liver metabolism in patients with advanced CHC. In patients with hepatitis C‐related liver cirrhosis, liver tissue miR‐10a levels were significantly associated with hepatic reserve, fibrosis markers, esophageal varix complications, and hepatitis C‐related hepatocellular carcinoma recurrence. Conclusion: MiRNA‐10a is involved in abnormal liver metabolism in cirrhotic liver through down‐regulation of the expression of the circadian rhythm gene Bmal1. Therefore, miR‐10a is a possible useful biomarker for estimating the prognosis of liver cirrhosis.