PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398-415).

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

b-AP15 [(3E,5E)-3,5-bis[(4-nitrophenyl)methylidene]-1-(prop-2-enoyl)piperidin-4-one] is a small molecule inhibitor of the ubiquitin specific peptidase (USP) 14/ubiquitin carboxyl-terminal hydrolase (UCH) L5 deubiquitinases of the 19S proteasome that shows antitumor activity in a number of tumor models, including multiple myeloma. b-AP15 contains an α,β-unsaturated carbonyl unit that is likely to react with intracellular nucleophiles such as cysteine thiolates by Michael addition. We found that binding of b-AP15 to USP14 is partially reversible, and that inhibition of proteasome function is reversible in cells. Despite reversible binding, tumor cells are rapidly committed to apoptosis/cell death after exposure to b-AP15. We show that b-AP15 is rapidly taken up from the medium and enriched in cells. Enrichment provides an explanation of the stronger potency of the compound in cellular assays compared with in vitro biochemical assays. Cellular uptake was impaired by 30-minute pretreatment of cells with low concentrations of N-ethylmaleimide (10 µM), suggesting that enrichment was thiol dependent. We report that in addition to inhibition of deubiquitinases, b-AP15 inhibits the selenoprotein thioredoxin reductase (TrxR). Whereas proteasome inhibition was closely associated with cell death induction, inhibition of TrxR was not. TrxR inhibition is, however, likely to contribute to triggering of oxidative stress observed with b-AP15. Furthermore, we present structure-activity, in vivo pharmacokinetic, and hepatocyte metabolism data for b-AP15. We conclude that the strong enrichment of b-AP15 in cells and a rapid commitment to apoptosis/cell death are factors that likely contribute to the strong antitumor activity of this compound.

Previous studies have suggested that hepatitis B virus (HBV) blocks expression of the alpha interferon (IFN-a)-inducible myeloid differential primary response protein (MyD88) gene. To study the molecular mechanism(s) of the inhibition of MyD88 expression by HBV, MyD88 promoter reporter plasmids and vectors expressing different HBV viral proteins were constructed. Co-transfection experiments showed that IFN-induced MyD88 promoter activity was inhibited by HBV polymerase expression in a dose-dependent manner and that the terminal protein (TP) domain of HBV polymerase was responsible for this antagonistic activity. Analysis of site mutants showed that the region targeted by the polymerase protein contained the signal transducer and activator of transcription (Stat) binding site. Chromatin immunoprecipitation analysis showed that the IFN-induced DNA-binding activity of Stat1 was affected. Further study demonstrated that the HBV polymerase protein inhibited the Stat1 nuclear translocation induced by IFN-a, but did not induce Stat1 degradation nor interfere with its phosphorylation. In addition, HBV polymerase could inhibit the transcriptional activity of other IFN-stimulated response element-driven promoters and the expression of interferon-stimulated genes (ISGs), such as Stat1 and ISG15. In summary, these results indicate that HBV polymerase is a general inhibitor of IFN signalling and can inhibit IFN-inducible MyD88 expression by inhibiting the activity of the MyD88 promoter through blocking the nuclear translocation of Stat1.

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox)-exo had enhanced proteolytic activity compared with HepAD38 (dox)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.

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Hepatitis C virus non-structural protein NS5A plays an important role in viral replication and various cellular events. To gain further insight into the function of NS5A, we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. FKBP38, a 38 kDa immunosuppressant FK506-binding protein, was identified and its interaction with NS5A was confirmed by both in vitro and in vivo. The interaction was mapped to the amino acids 148-236 of NS5A containing a BH domain (Bcl-2 homology domain). Besides, both NS5A and FKBP38 were found to localize in mitochondria and endoplasmic reticulum. Moreover, NS5A stably expressing Huh7 hepatoma cells showed more resistance to apoptosis and such inhibition of apoptosis could specifically be abrogated by depletion of FKBP38 using RNA interference. These results indicate that HCV NS5A inhibits apoptosis through interaction with FKBP38.

SARS coronavirus (SARS-CoV) is known to efficiently suppress the induction of antiviral type I interferons (IFN-a/b) in non-lymphatic cells through inhibition of the transcription factor IRF-3.Plasmacytoid dendritic cells, in contrast, respond to infection with production of high levels of IFNs. Here, we show that pretreatment of non-lymphatic cells with small amounts of IFN-a (IFN priming) partially overturns the block in IFN induction imposed by SARS-CoV. IFN priming combined with SARS-CoV infection substantially induced genes for IFN induction, IFN signalling, antiviral effector proteins, ubiquitination and ISGylation, antigen presentation and other cytokines and chemokines, whereas each individual treatment had no major effect. Curiously, however, despite this typical IFN response, neither IRF-3 nor IRF-7 was transported to the nucleus as a sign of activation. Taken together, our results suggest that (i) IFN, as it is produced by plasmacytoid dendritic cells, could enable tissue cells to launch a host response to SARSCoV, (ii) IRF-3 and IRF-7 may be active at subdetectable levels, and (iii) SARS-CoV does not activate IRF-7. INTRODUCTIONSARS coronavirus (SARS-CoV) is the causative agent of severe acute respiratory syndrome (SARS), a life-threatening human disease that has recently emerged in China (Drosten et al., 2003;Ksiazek et al., 2003;Kuiken et al., 2003;Peiris et al., 2003b). SARS-CoV causes high fever, myalgia, dry cough and lymphopenia, and around 30 % of patients develop an atypical pneumonia (Denison, 2004;Peiris et al., 2004). The worldwide epidemic in spring 2003 resulted in over 8000 cases with 774 deaths (WHO, 2004). SARS-CoV is known to be sensitive to the antiviral action of type I interferons (IFN-a/b) both in cell culture and in vivo (Cinatl et al., 2003;Haagmans et al., 2004;Stroher et al., 2004). IFNs are synthesized and secreted by infected cells and stimulate expression of potent antiviral proteins (Sadler & Williams, 2008;Samuel, 2001). IFN induction in tissue cells occurs mainly by an intracellular pathway. Hallmark molecules of virus infection, such as doublestranded RNA (dsRNA) or 59-triphosphorylated singlestranded RNA, are recognized by cellular receptors such as RIG-I and MDA5, and activate a signalling chain resulting in phosphorylation of the transcription factor IRF-3 (Pichlmair & Reis e Sousa, 2007;Yoneyama & Fujita, 2008). IRF-3 is a member of the IFN-regulatory factor (IRF) family and plays a central role in the transactivation of the IFN-b promoter (Hiscott, 2007). Phosphorylated IRF-3 homodimerizes and moves into the nucleus, where it initiates IFN-b mRNA synthesis (Suhara et al., 2002;Weaver et al., 1998). This first-wave IFN triggers expression of a related factor, IRF-7, which is present only in low amounts in unstimulated fibroblasts (Honda et al., 2005). IRF-7 can be activated by the same pathway as IRF-3 et al., 1991) by digestion with ClaI and XhoI restriction endonucleases to obtain the construct pCAGGs-hIRF7A. The green fluorescent protein (GFP) fusion construc...