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Background & Aims: A 3-dimensional (3D) culture system for immortalized human hepatocytes (HuS-E/2 cells) recently was shown to support the lifecycle of blood-borne hepatitis C virus (HCV). We used this system to identify proteins that are active during the HCV lifecycle under 3D culture conditions. Methods: We compared gene expression profiles of HuS-E/2 cells cultured under 2-dimensional and 3D conditions. We identified signaling pathways that were activated differentially in the cells, and analyzed their functions in the HCV lifecycle using a recombinant HCV-producing cell-culture system, with small interfering RNAs and chemical reagents. We investigated the effects of anti-HCV reagents that altered these signaling pathways in mice with humanized livers (carrying human hepatocytes). Results: Microarray analysis showed that cells cultured under 2-dimensional vs 3D conditions expressed different levels of messenger RNAs encoding prostaglandin synthases. Small interfering RNA–mediated knockdown of thromboxane A2 synthase (TXAS) and incubation of hepatocytes with a TXAS inhibitor showed that this enzyme is required for production of infectious HCV, but does not affect replication of the HCV genome or particle release. The TXAS inhibitor and a prostaglandin I2 receptor agonist, which has effects that are opposite those of thromboxane A2, reduced serum levels of HCV and inhibited the infection of human hepatocytes by blood-borne HCV in mice. Conclusions: An inhibitor of the prostaglandin synthase TXAS inhibits production of infectious HCV particles in cultured hepatocytes and HCV infection of hepatocytes in mice with humanized livers. It therefore might be therapeutic for HCV infection. [Copyright &y& Elsevier]

A cell culture system that allows the reproduction of the hepatitis B virus (HBV) life cycle is indispensable to exploring novel anti-HBV agents. To establish the screening system for anti-HBV agents, we exploited the high affinity and bright luminescence (HiBiT) tag and comprehensively explored the regions in the HBV genome where the HiBiT tag could be inserted. The plasmids for the HiBiT-tagged HBV molecular clones with a 1.38-fold HBV genome length were prepared. The HiBiT tag was inserted into five regions: preS1, preS2, hepatitis B e (HBe), hepatitis B X (HBx), and hepatitis B polymerase (HB pol). HiBiT-tagged HBVs were obtained by transfecting the prepared plasmids into sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells, and their infectivity was evaluated in human primary hepatocytes and HepG2/NTCP cells. Among the evaluated viruses, the infection of HiBiT-tagged HBVs in the preS1 or the HB pol regions exhibited a time-dependent increase of the hepatitis B surface antigen (HBsAg) level after infection to HepG2/NTCP cells as well as human primary hepatocytes. Immunostaining of the hepatitis B core (HBc) antigen in infected cells confirmed these viruses are infectious to those cells. However, the time-dependent increase of the HiBiT signal was only detected after infection with the HiBiT-tagged HBV in the preS1 region. The inhibition of this HiBiT-tagged HBV infection in human primary hepatocytes and HepG2/NTCP cells by the preS1 peptide could be detected by measuring the HiBiT signal. The infection system with the HiBiT-tagged HBV in HepG2/NTCP cells facilitates easy, sensitive, and high-throughput screening of anti-HBV agents and will be a useful tool for assessing the viral life cycle and exploring antiviral agents., Importance: Hepatitis B virus (HBV) is the principal causative agent of chronic hepatitis. Despite the availability of vaccines in many countries, HBV infection has spread worldwide and caused chronic infection. In chronic hepatitis B patients, liver inflammation leads to cirrhosis, and the accumulation of viral genome integration into host chromosomes leads to the development of hepatocellular carcinoma. The currently available treatment strategy cannot expect the eradication of HBV. To explore novel anti-HBV agents, a cell culture system that can detect HBV infection easily is indispensable. In this study, we examined the regions in the HBV genome where the high affinity and bright luminescence (HiBiT) tag could be inserted and established an HBV infection system to monitor infection by measuring the HiBiT signal by infecting the HiBiT-tagged HBV in sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. This system can contribute to screening for novel anti-HBV agents.