A recombinant hepatitis B virus (HBV) expressing NanoLuc (NL) (HBV/NL) was produced by cotransfecting a plasmid containing a 1.2‐fold HBV genome carrying the NL gene with a plasmid bearing a packaging‐defective 1.2‐fold HBV genome into a human hepatoma cell line, HepG2. We found that NL activity in HBV/NL‐infected primary hepatocytes or sodium taurocholate cotransporting polypeptide‐transduced human hepatocyte‐derived cell lines increased linearly for several days after infection and was concordant with HBV RNA levels in the cells. Treatment of the virus‐infected cells with HBV inhibitors reduced NL activity in a dose‐dependent manner. Detection of HBV/NL infection, monitored by NL activity, was highly sensitive and less expensive than detection using the conventional method to evaluate HBV infection. In addition, because we also studied host factors, this system is applicable not only for studying the HBV life cycle, but also for exploring agent(s) that regulate HBV proliferation.

Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses.lncRNA | innate immunity | interferon | ISG

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV)is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1␣ (MIP-1␣), and MIP-1␤. Moreover, we found that this effect was mediated by IL-1␣, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) ␤ mRNA, and HSC-dependent IL-1␣ production contributed to the stimulation of C/EBP␤ target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBP␤ in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

The hepatitis C virus (HCV) is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using 17-allylaminogeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (Hsp90). Hsp90 is a molecular chaperone with a key role in stabilizing the conformation of many oncogenic signaling proteins. We examined the inhibitory effects of 17-AAG on HCV replication in an HCV replicon cell culture system. In HCV replicon cells treated with 17-AAG, we found that HCV RNA replication was suppressed in a dose-dependent manner, and interestingly, the only HCV protein degraded in these cells was NS3 (nonstructural protein 3). Immunoprecipitation experiments showed that NS3 directly interacted with Hsp90, as did proteins expressed from ⌬NS3 protease expression vectors. These results suggest that the suppression of HCV RNA replication is due to the destabilization of NS3 in disruption of the Hsp90 chaperone complex by 17-AAG. Infection by the hepatitis C virus (HCV)2 is a major public health problem, with 170 million chronically infected people worldwide (1, 2). The current treatment by combined interferon-ribavirin therapy fails to cure the infection in 30 -50% of cases (3, 4), particularly those with HCV genotypes 1 and 2. Chronic infection with HCV results in liver cirrhosis and can lead to hepatocellular carcinoma (5, 6). Although an effective combined interferon-␣-ribavirin therapy is available for about 50% of the patients with HCV, better therapies are needed, and preventative vaccines have not yet been developed.HCV is a member of the Flaviviridae family and has a positive strand RNA genome (7, 8) that encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, E1 (envelope 1), E2, p7, NS2 (nonstructural protein 2), NS3, NS4A, NS4B, NS5A, and NS5B (9, 10). NS2 and the amino terminus of NS3 comprise the NS2-3 protease responsible for cleavage between NS2 and NS3 (9, 11), whereas NS3 is a multifunctional protein consisting of an amino-terminal protease domain required for processing NS3 to NS5B (12, 13). NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer (14 -17), and the hydrophobic protein NS4B induces the formation of a cytoplasmic vesicular structure, designated the membranous web, which is likely to contain the replication complex of HCV (18,19). NS5A is a phosphoprotein that appears to play an important role in viral replication (20 -23), and NS5B is the RNA-dependent RNA polymerase of HCV (24, 25). The 3Ј-untranslated region consists of a short variable sequence, a poly(U)-poly(UC) tract, and a highly conserved X region and is critical for HCV RNA replication and HCV infection (26 -29).Hsp90 (heat-shock protein 90) is a molecular chaperone that plays a key role in the conformational maturation of many cellular proteins. Hsp90 normally functions in association with other co-chaperone proteins, which together play an important role in folding newly ...

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.