Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

Wnt/␤-catenin signaling contributes to diverse cellular functions, such as Drosophila wing development and colon carcinogenesis. Recently, stabilizing mutations of ␤-catenin, a hallmark of Wnt signaling, were documented in significant numbers of primary hepatocellular carcinomas (HCC). However, whether the ␤-catenin mutation leads to the activation of Wnt/␤-catenin signaling in hepatoma cells has not been established. We found that Wnt/ ␤-catenin signaling could be activated by ectopic expression of Wnt-1 in some hepatoma cells, such as Hep3B and PLC/PRF/5 cells, but not in others, such as Huh7 and Chang cells. Importantly, we noted that the former were derived from hepatitis B virus (HBV)-infected livers, whereas the latter were derived from HBV-negative livers. It was then speculated that HBx, a viral regulatory protein of HBV, is involved in activating Wnt/␤-catenin signaling in hepatoma cells. In agreement with this notion, ectopic expression of HBx along with Wnt-1 activated Wnt/␤-catenin signaling in Huh7 cells by stabilizing cytoplasmic ␤-catenin. Further, we showed that such stabilization of ␤-catenin by HBx was achieved by suppressing glycogen synthase kinase 3 activity via the activation of Src kinase. In conclusion, the data suggest that Wnt-1 is necessary but insufficient to activate Wnt/␤-catenin signaling in hepatoma cells and the enhanced stabilization of ␤-catenin by HBx, in addition to Wnt-1, is essential for the activation of Wnt/␤-catenin signaling in hepatoma cells. (HEPATOLOGY 2004;

Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKε activity by disrupting the interaction between IKKε and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKε activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.

Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.Viruses rely on host factors to complete their life cycles. These factors facilitate many steps of the viral life cycle, including entry, uncoating, genome replication, viral assembly, and virus release (3, 13). Recently, some host factors that contribute to the life cycles of some clinically important human viruses were identified by full-genome small interfering RNA knockdown experiments (7,14,31). For instance, nearly 300 host factors that contribute to human immunodeficiency virus (HIV) infection were identified (7). Yet, little is known about host factors that contribute to the genome replication of hepatitis B virus (HBV).HBV, the prototypical member of the hepadnavirus family, is a major cause of liver disease worldwide (34). HBV-mediated disease manifestations range from acute and chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV contains a DNA genome, the replication of the genome occurs by reverse transcription of the pregenomic RNA (pgRNA) template. HBV polymerase (Pol), or reverse transcriptase, acts as an RNA binding protein by specifically recognizing an RNA stem-loop structure called the 5Ј ε encapsidation signal (5Ј ε), and this interaction is required for pgRNA encapsidation (5, 15, 17). Viral reverse transcription occurs entirely within nucleocapsids following encapsidation. HBV reverse transcription has two steps for DNA synthesis: (i) minus-strand DNA synthesis and (ii) plus-strand DNA synthesis. During the first step, the pgRNA is converted i...

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.

Drug repositioning represents an effective way to control the current COVID‐19 pandemic. Previously, we identified 24 FDA‐approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. The comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC 50 = 0.0022 µM).

HBx, a small regulatory protein of hepatitis B virus, plays an important role in stimulating viral genome replication. HBx was shown to be associated with diverse subcellular locations, such as the nucleus, cytoplasm and mitochondria. Some studies have linked the stimulation of genome replication by HBx to its cytoplasmic function, while other reports have attributed this function to its nuclear component. To clarify this discrepancy, we measured viral genome replication by complementing an HBx-null replicon in two different ways: by (i) co-transfecting with an increasing amount of HBx expression plasmid and (ii) co-transfecting with re-targeted variants of HBx that are confined to either the nucleus or the cytoplasm due to either the nuclear localization signal (NLS) or the nuclear export signal (NES) tags, respectively. Intriguingly, immunostaining analysis indicated that the subcellular localization of HBx is primarily influenced by its abundance; HBx is confined to the nucleus at low levels but is usually detected in the cytoplasm at high levels. Importantly, HBx, whether re-targeted by either the NLS or NES tag, stimulates viral genome replication to a level comparable to that of the wild-type. Furthermore, similar to the wild-type, the stimulation of viral genome replication by the re-targeted HBx occurred at the transcription level. Thus, we concluded that the stimulation of viral genome replication by HBx is linked to both nuclear and cytoplasmic HBx, although the underlying mechanism of stimulation most likely differs.

DDX3 is a member of the DEAD-box RNA helicase family, involved in mRNA metabolism, including transcription, splicing, and translation. We previously identified DDX3 as a hepatitis B virus (HBV) polymerase (Pol) binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at the posttranscriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed a diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracyclineinducible HBV-producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBVsusceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription. IMPORTANCE Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution, viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5␣ and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to the case with retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3DEAD box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism, such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription. C hronic hepatitis B virus (HBV) infection represents a major public health burden, affecting more than 300 million individuals worldwide, and carries a high risk for developing cirrhosis and hepatocellular carcinoma (HCC) (1). HBV virions contain a small, partially double-stranded circular DNA genome of 3.2 kb in length. Although it is a DNA virus, HBV replicates its DNA genome via reverse transcription. Upon infection, the virion DNA is converted into covalently closed circular DNA (cccDNA), which then serves as the template for viral transcription (2). Among the viral transcripts, only pregenomic RNA (pgRNA), 3.5 kb in length, is selectively packaged into nucleocapsids along with HBV polymerase (Pol). Inside the nucleocapsid, the pgRNA is reverse transcribed by HBV Pol to yield relaxed circular (RC) DNA. These mature RC DNA-containin...