Mitochondrial HIGD1A inhibits hepatitis B virus transcription and replication through the cellular PNKD-NF-κB-NR2F1 nexus.

Hepatitis B Virus (HBV) replication has been reported to be restricted by the intrahepatic host restriction factors and antiviral signaling pathways. The intracellular mechanisms underlying the significant viremia difference among different phases of the natural history chronic HBV infection remain elusive. We herein report that the hypoxia-induced gene domain protein-1a (HIGD1A) was highly expressed in the liver of inactive HBV carriers with low viremia. Ectopic expression of HIGD1A in hepatocyte-derived cells significantly inhibited HBV transcription and replication in a dose-dependent manner, while silence of HIGD1A promoted HBV gene expression and replication. Similar results were also observed in both de novo HBV-infected cell culture model and HBV persistence mouse model. Mechanistically, HIGD1A is located on the mitochondrial inner membrane and activates nuclear factor kappa B (NF-κB) signaling pathway through binding to paroxysmal nonkinesigenic dyskinesia (PNKD), which further enhances the expression of a transcription factor NR2F1 to inhibit HBV transcription and replication. Consistently, knockdown of PNKD or NR2F1 and blockage of NF-κB signaling pathway abrogated the inhibitory effect of HIGD1A on HBV replication. Mitochondrial HIGD1A exploits the PNKD-NF-κB-NR2F1 nexus to act as a host restriction factor of HBV infection. Our study thus shed new lights on the regulation of HBV by hypoxia-related genes and related antiviral strategies.

Endoplasmic reticulum stress promotes HBV production by enhancing use of the autophagosome/multivesicular body axis.

BACKGROUND AND AIMS: HBV infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion and examined the underlying mechanisms. APPROACH AND RESULTS: Protein disulfide isomerase (an ER marker), microtubule-associated protein 1 light chain 3 beta (an autophagosome [AP] marker), and sequestosome-1 (a typical cargo for autophagic degradation) expression were tested in liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased AP formation, resulting in enhanced HBV replication and secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBsAg localization in this compartment, causing HBsAg/SVPs to accumulate in the ER. Thus, TM-induced AP formation serves as an alternative pathway for HBsAg/SVP trafficking. Importantly, TM inhibited AP-lysosome fusion, accompanied by enhanced AP/late endosome (LE)/multivesicular body fusion, to release HBsAg/SVPs through, or along with, exosome release. Notably, TM treatment inhibited HBsAg glycosylation, resulting in impairment of HBV virions' envelopment and secretion, but it was not critical for HBsAg/SVP trafficking in our cell systems. CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the AP-LE/MVB axis.

A systematic comparison reveals dynamic differences in early adaptive immune responses of acute-resolving versus chronic HBV replication.

Chronic hepatitis B virus (HBV) infection has been characterized by lack of effective adaptive immune responses which are vital for the viral clearance. However, very little is known about the dynamics of adaptive immune responses during the early phase of chronic HBV infection especially in spleen and liver. Here, we used the hydrodynamic injection (HDI) mouse model to kinetically characterize differences in the features of adaptive immunity, including the frequencies, phenotypes and function of antigen-presenting cells and T cells in the spleen, peripheral blood mononuclear cells (PBMCs) and liver, of chronic versus acute-resolving HBV replication (AR). We found that mice with AR mice and mice with chronic HBV replication (CH) mice showed early splenomegaly accompanied by T cell expansion in spleen but not in liver after HDI. Interestingly, the early and continuous increase in HBV-specific CD8+ T cells in spleen of CH mice was comparable to that in the AR mice. However, the splenic T cells of CH mice showed no activation phenotype compared with those in AR mice. Besides, increases in activated effector CD8+ T cells in PBMCs and liver at later time points were only observed in AR mice but not CH mice. CH mice also showed insufficient expansion of dendritic cells (DCs) in spleen and increased programmed death-1 expression in DCs of the liver compared to AR mice. The adoptive transfer of total splenocytes or splenic CD8+ T cells of AR mice to CH mice demonstrated that their ability to break HBV tolerance varies at different stages of HBV clearance. Moreover, the adoptive transfer of splenocytes from AR mice induce functional activation of endogenous HBV-specific CD8+ T cells of CH mice. Our results suggest that early T cell priming and expansion initially happens in the periphery after HBV antigen exposure in acute-resolving and chronic replication. The paucity of T cell activation, and subsequent migration and liver infiltration is a key feature of the adaptive immune responses during the early phase of CH, which is probably caused by the dysfunction of DCs.

Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. METHODS: Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. RESULTS: Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. CONCLUSION: HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

Aborted infection of human sodium taurocholate cotransporting polypeptide (hNTCP) expressing woodchuck hepatocytes with hepatitis B virus (HBV).

Due to the limited host range of HBV, research progress has been hindered by the absence of a suitable animal model. The natural history of woodchuck hepatitis virus (WHV) infection in woodchuck closely mirrors that of HBV infection in human, making this species a promising candidate for establishing both in vivo and in vitro HBV infection models. Therefore, this animal may be a valuable species to evaluate HBV vaccines and anti-HBV drugs. A significant milestone in HBV and hepatitis D virus (HDV) infection is the discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the functional receptor. In an effort to enhance susceptibility to HBV infection, we introduced hNTCP into the woodchuck hepatocytes by multiple approaches including transduction of vLentivirus-hNTCP in woodchuck hepatocytes, transfection of p-lentivirus-hNTCP-eGFP plasmids into these cells, as well as transduction of vAdenovirus-hNTCP-eGFP. Encouragingly, our findings demonstrated the successful introduction of hNTCP into woodchuck hepatocytes. However, it was observed that these hNTCP-expressing hepatocytes were only susceptible to HDV infection but not HBV. This suggests the presence of additional crucial factors mediating early-stage HBV infection that are subject to stringent species-specific restrictions.

Contribution of T- and B-cell intrinsic toll-like receptors to the adaptive immune response in viral infectious diseases.

Toll-like receptors (TLRs) comprise a class of highly conserved molecules that recognize pathogen-associated molecular patterns and play a vital role in host defense against multiple viral infectious diseases. Although TLRs are highly expressed on innate immune cells and play indirect roles in regulating antiviral adaptive immune responses, intrinsic expression of TLRs in adaptive immune cells, including T cells and B cells, cannot be ignored. TLRs expressed in CD4 + and CD8 + T cells play roles in enhancing TCR signal-induced T-cell activation, proliferation, function, and survival, serving as costimulatory molecules. Gene knockout of TLR signaling molecules has been shown to diminish antiviral adaptive immune responses and affect viral clearance in multiple viral infectious animal models. These results have highlighted the critical role of TLRs in the long-term immunological control of viral infection. This review summarizes the expression and function of TLR signaling pathways in T and B cells, focusing on the in vitro and vivo mechanisms and effects of intrinsic TLR signaling in regulating T- and B-cell responses during viral infection. The potential clinical use of TLR-based immune regulatory drugs for viral infectious diseases is also explored.

Prophylactic and therapeutic HBV vaccination by an HBs-expressing cytomegalovirus vector lacking an interferon antagonist in mice.

Cytomegalovirus (CMV)-based vaccines show promising effects against chronic infections in nonhuman primates. Therefore, we examined the potential of hepatitis B virus (HBV) vaccines based on mouse CMV (MCMV) vectors expressing the small HBsAg. Immunological consequences of vaccine virus attenuation were addressed by either replacing the dispensable gene m157 ("MCMV-HBsȍ) or the gene M27 ("ΔM27-HBs"), the latter encodes a potent IFN antagonist targeting the transcription factor STAT2. M27 was chosen, since human CMV encodes an analogous gene product, which also induced proteasomal STAT2 degradation by exploiting Cullin RING ubiquitin ligases. Vaccinated mice were challenged with HBV through hydrodynamic injection. MCMV-HBs and ΔM27-HBs vaccination achieved accelerated HBV clearance in serum and liver as well as robust HBV-specific CD8+ T-cell responses. When we explored the therapeutic potential of MCMV-based vaccines, especially the combination of ΔM27-HBs prime and DNA boost vaccination resulted in increased intrahepatic HBs-specific CD8+ T-cell responses and HBV clearance in persistently infected mice. Our results demonstrated that vaccines based on a replication competent MCMV attenuated through the deletion of an IFN antagonist targeting STAT2 elicit robust anti-HBV immune responses and mediate HBV clearance in mice in prophylactic and therapeutic immunization regimes.

Simultaneous or Prior Activation of Intrahepatic Type I Interferon Signaling Leads to Hepatitis B Virus Persistence in a Mouse Model.

It remains controversial how interferon (IFN) response contributes to hepatitis B virus (HBV) control and pathogenesis. A previous study identified that hydrodynamic injection (HI) of type I IFN (IFN-I) inducer polyinosinic-poly(C) [poly(I·C)] leads to HBV clearance in a chronic HBV mouse model. However, recent studies have suggested that premature IFN-I activation in the liver may facilitate HBV persistence. In the present study, we investigated how the early IFN-I response induces an immunosuppressive signaling cascade and thus causes HBV persistence. We performed HI of the plasmid adeno-associated virus (pAAV)/HBV1.2 into adult BALB/c mice to establish an adult acute HBV replication model. Activation of the IFN-I signaling pathway following poly(I·C) stimulation or murine cytomegalovirus (MCMV) infection resulted in subsequent HBV persistence. HI of poly(I·C) with the pAAV/HBV1.2 plasmid resulted in not only the production of IFN-I and the anti-inflammatory cytokine interleukin-10 (IL-10) but also the expansion of intrahepatic regulatory T cells (Tregs), Kupffer cells (KCs), and myeloid-derived suppressor cells (MDSCs), all of which impaired the T cell response. However, when poly(I·C) was injected at day 14 after the HBV plasmid injection, it significantly enhanced HBV-specific T cell responses. In addition, interferon-alpha/beta receptor (IFNAR) blockade rescued T cell response by downregulating IL-10 expression and decreasing Treg and KC expansion. Consistently, Treg depletion or IL-10 blockade also controlled HBV replication. IMPORTANCE IFN-I plays a double-edged sword role during chronic HBV infection. Here, we identified that application of IFN-I at different time points causes contrast outcomes. Activation of the IFN-I pathway before HBV replication induces an immunosuppressive signaling cascade in the liver and consequently caused HBV persistence, while IFN-I activation post HBV infection enhances HBV-specific T cell responses and thus promotes HBV clearance. This result provided an important clue to the mechanism of HBV persistence in adult individuals.

Functional Comparison of Interferon-α Subtypes Reveals Potent Hepatitis B Virus Suppression by a Concerted Action of Interferon-α and Interferon-γ Signaling.

BACKGROUND AND AIMS: Interferon (IFN)-α, composed of numerous subtypes, plays a crucial role in immune defense. As the most studied subtype, IFN-α2 has been used for treating chronic hepatitis B virus (HBV) infection, with advantages of finite treatment duration and sustained virologic response, but its efficacy remains relatively low. This study aimed to screen for IFN-α subtypes with the highest anti-HBV potency and to characterize mechanisms of IFN-α-mediated HBV restriction. APPROACH AND RESULTS: Using cell culture-based HBV infection systems and a human-liver chimeric mouse model, IFN-α subtype-mediated antiviral response and signaling activation were comprehensively analyzed. IFN-α14 was identified as the most effective subtype in suppression of HBV covalently closed circular DNA transcription and HBV e antigen/HBV surface antigen production, with median inhibitory concentration values approximately 100-fold lower than those of the conventional IFN-α2. IFN-α14 alone elicited IFN-α and IFN-γ signaling crosstalk in a manner similar to the combined use of IFN-α2 and IFN-γ, inducing multiple potent antiviral effectors, which synergistically restricted HBV replication. Guanylate binding protein 5, one of the most differentially expressed genes between IFN-α14-treated and IFN-α2-treated liver cells, was identified as an HBV restriction factor. A strong IFN-α-IFN-α receptor subunit 1 interaction determines the anti-HBV activity of IFN-α. The in vivo anti-HBV activity of IFN-α14 and treatment-related transcriptional patterns were further confirmed, and few adverse effects were observed. CONCLUSIONS: A concerted IFN-α and IFN-γ response in liver, which could be efficiently elicited by IFN-α subtype 14, is associated with potent HBV suppression. These data deepen the understanding of the divergent activities of IFN-α subtypes and the mechanism underlying the synergism between IFN-α and IFN-γ signaling, with implications for improved IFN therapy and HBV curative strategies.

Inducible LGALS3BP/90K activates antiviral innate immune responses by targeting TRAF6 and TRAF3 complex.

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production.

Major Vault Protein Promotes Hepatocellular Carcinoma Through Targeting Interferon Regulatory Factor 2 and Decreasing p53 Activity.

BACKGROUND AND AIMS: Major vault protein (MVP) is up-regulated during infections with hepatitis B virus (HBV) and hepatitis C virus (HCV). Here, we found that MVP deficiency inhibited hepatocellular carcinoma (HCC) development induced by diethylnitrosamine, hepatitis B X protein, and HCV core. APPROACH AND RESULTS: Forced MVP expression was sufficient to induce HCC in mice. Mechanistic studies demonstrate that the ubiquitin ligase human double minute 2 (HDM2) forms mutual exclusive complexes either with interferon regulatory factor 2 (IRF2) or with p53. In the presence of MVP, HDM2 is liberated from IRF2, leading to the ubiquitination of the tumor suppressor p53. Mouse xenograft models showed that HBV and HCV promote carcinogenesis through MVP induction, resulting in a loss of p53 mediated by HDM2. Analyses of clinical samples from chronic hepatitis B, liver cirrhosis, and HCC revealed that MVP up-regulation correlates with several hallmarks of malignancy and associates with poor overall survival. CONCLUSIONS: Taken together, through the sequestration of IRF2, MVP promotes an HDM2-dependent loss of p53 that promotes HCC development.

Hepatitis B Virus Particles Activate Toll-Like Receptor 2 Signaling Initially Upon Infection of Primary Human Hepatocytes.

BACKGROUND AND AIMS: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo. APPROACH AND RESULTS: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen. CONCLUSIONS: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV.

O-GlcNAcylation modulates HBV replication through regulating cellular autophagy at multiple levels.

O-GlcNAcylation is a form of posttranslational modification, and serves various functions, including modulation of location, stability, and activity for the modified proteins. O-linked-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies the cellular proteins with O-GlcNAc moiety. Early studies reported that the decreased O-GlcNAcylation regulates cellular autophagy, a process relevant for hepatitis B virus replication (HBV) and assembly. Therefore, we addressed the question how O-GlcNAcylation regulates cellular autophagy and HBV replication. Inhibition of OGT activity with a small molecule inhibitor OSMI-1 or silencing OGT significantly enhanced HBV replication and HBsAg production in hepatoma cells and primary human hepatocytes (PHHs). Western blotting analysis showed that inhibition of O-GlcNAcylation-induced endoplasmic reticulum (ER) stress and cellular autophagy, two processes subsequently leading to enhanced HBV replication. Importantly, the numbers of autophagosomes and the levels of autophagic markers LC3-II and SQSTM1/p62 in hepatoma cells were elevated after inhibition of O-GlcNAcylation. Further analysis revealed that inhibition of O-GlcNAcylation blocked autophagosome-lysosome fusion and thereby prevented autophagic degradation of HBV virions and proteins. Moreover, OSMI-1 further promoted HBV replication by inducing autophagosome formation via inhibiting the O-GlcNAcylation of Akt and mTOR. In conclusion, decreased O-GlcNAcylation enhanced HBV replication through increasing autophagosome formation at multiple levels, including triggering ER-stress, Akt/mTOR inhibition, and blockade of autophagosome-lysosome fusion.

AMPK and Akt/mTOR signalling pathways participate in glucose-mediated regulation of hepatitis B virus replication and cellular autophagy.

A growing consensus indicates that host metabolism plays a vital role in viral infections. Hepatitis B virus (HBV) infection occurs in hepatocytes with active glucose metabolism and may be regulated by cellular metabolism. We addressed the question whether and how glucose regulates HBV replication in hepatocytes. The low glucose concentration at 5 mM significantly promoted HBV replication via enhanced transcription and autophagy when compared with higher glucose concentrations (10 and 25 mM). At low glucose concentration, AMPK activity was increased and led to ULK1 phosphorylation at Ser 555 and LC3-II accumulation. By contrast, the mTOR pathway was activated by high glucose concentrations, resulting in reduced HBV replication. mTOR inhibition by rapamycin reversed negative effects of high glucose concentrations on HBV replication, suggesting that low glucose concentration promotes HBV replication by stimulating the AMPK/mTOR-ULK1-autophagy axis. Consistently, we found that glucose transporters inhibition using phloretin also enhanced HBV replication via increased AMPK/mTOR-ULK1-induced autophagy. Surprisingly, the glucose analogue 2-deoxy-D-glucose reduced HBV replication through activating the Akt/mTOR signalling pathway also at the low glucose concentrations. Our study reveals that glucose is an important factor for the HBV life cycle by regulating HBV transcription and posttranscriptional steps of HBV replication via cellular autophagy.

The impact of hepatitis B surface antigen on natural killer cells in patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: During chronic hepatitis B virus (HBV) infection, suppressed functionality of natural killer (NK) cells might contribute to HBV persistence but the underlying mechanisms remain elusive. A peculiar feature of HBV is the secretion of large amount of hepatitis B surface antigen (HBsAg). However, the effect of HBsAg quantities on NK cells is unclear. The aim was to determine the effects of HBsAg quantities on NK cell functionality in patients with chronic hepatitis B (CHB). METHODS: Eighty CHB patients were included and categorized into four groups based on their HBsAg levels. As a control, 30 healthy donors were enrolled. NK cell frequency, phenotype and function were assessed using flow cytometry and correlated with HBsAg levels and liver enzymes. RESULTS: Compared to the healthy controls, a reshaping of NK cell pool towards more CD56bright NK cells was observed during CHB infection. Importantly, NK cells in patients with low HBsAg levels (<100 IU/mL) displayed an activated phenotype with increased expression of activation makers CD38, granzyme B and proliferation marker Ki-67 while presenting with defective functional responses (MIP-1ß, CD107a) at the same time. Furthermore, NK cell activation was negatively correlated with patient HBsAg levels while NK function correlated with patient age. CONCLUSIONS: The differential regulation of NK cell phenotype and function suggests that activation of NK cells in patients with low serum HBsAg levels may contribute to HBV clearance.

TLR2 Stimulation Increases Cellular Metabolism in CD8+ T Cells and Thereby Enhances CD8+ T Cell Activation, Function, and Antiviral Activity.

TLR2 serves as a costimulatory molecule on activated T cells. However, it is unknown how the functionality and antiviral activity of CD8+ T cells are modulated by direct TLR2 signaling. In this study, we looked at the TLR2-mediated enhancement of TCR-driven CD8+ T cell activation in vitro and in woodchuck hepatitis virus transgenic mice. In vitro stimulation of CD8+ T cells purified from C57BL/6 mice showed that TLR2 agonist Pam3CSK4 directly enhanced the TCR-dependent CD8+ T cell activation. Transcriptome analysis revealed that TLR2 signaling increased expression of bioenergy metabolism-related genes in CD8+ T cells, such as IRF4, leading to improved glycolysis and glutaminolysis. This was associated with the upregulation of genes related to immune regulation and functions such as T-bet and IFN-γ. Glycolysis and glutaminolysis were in turn essential for the TLR2-mediated enhancement of T cell activation. Administration of TLR2 agonist Pam3CSK4 promoted the expansion and functionality of vaccine-primed, Ag-specific CD8+ T cells in both wild type and transgenic mice and improved viral suppression. Thus, TLR2 could promote CD8+ T cell immunity through regulating the energy metabolism.

TLR5 activation in hepatocytes alleviates the functional suppression of intrahepatic CD8+ T cells.

The liver is an immune-privileged organ with a tolerogenic environment for maintaining liver homeostasis. This hepatic tolerance limits the intrahepatic CD8+ T-cell response for eliminating infections. The tolerant microenvironment in the liver is orchestrated by liver-specific immunoregulatory cells that can be functionally regulated by pathogen-associated molecular patterns (PAMPs). Here, we report that flagellin, a key PAMP of gut bacteria, modulates the intrahepatic CD8+ T-cell response by activating the TLR5 signalling pathway of hepatocytes. We found that mice treated with Salmonella-derived recombinant flagellin (SF) by hydrodynamic injection had a significantly elevated IFN-γ production by the intrahepatic lymphocytes in 7 days after injection. This was correlated with a reduced immune suppressive effect of primary mouse hepatocytes (PMHs) in comparison with that of PMHs from mock-injected control mice. In vitro co-culture of SF-treated PMHs with splenocytes revealed that hepatocyte-induced immune suppression is alleviated through activation of the TLR5 but not the NLRC4 signalling pathway, leading to improved activation and function of CD8+ T cells during anti-CD3 stimulation or antigen-specific activation. In an acute HBV replication mouse model established by co-administration of SF together with an HBV-replicating plasmid by hydrodynamic injection, SF significantly enhanced the intrahepatic HBV-specific CD8+ T-cell response against HBV surface antigen. Our results clearly showed that flagellin plays a role in modulating the intrahepatic CD8+ T-cell response by activating the TLR5 pathway in PMHs, which suggests a potential role for gut bacteria in regulating liver immunity.

RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element.

Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

Epigenetic Modification Is Regulated by the Interaction of Influenza A Virus Nonstructural Protein 1 with the De Novo DNA Methyltransferase DNMT3B and Subsequent Transport to the Cytoplasm for K48-Linked Polyubiquitination.

The influenza virus nonstructural protein 1 (NS1) is a nonstructural protein that plays a major role in antagonizing host interferon responses during infection. However, a clear role for the NS1 protein in epigenetic modification has not been established. In this study, NS1 was found to regulate the expression of some key regulators of JAK-STAT signaling by inhibiting the DNA methylation of their promoters. Furthermore, DNA methyltransferase 3B (DNMT3B) is responsible for this process. Upon investigating the mechanisms underlying this event, NS1 was found to interact with DNMT3B but not DNMT3A, leading to the dissociation of DNMT3B from the promoters of the corresponding genes. In addition, the interaction between NS1 and DNMT3B changed the localization of DNMT3B from the nucleus to the cytosol, resulting in K48-linked ubiquitination and degradation of DNMT3B in the cytosol. We conclude that NS1 interacts with DNMT3B and changes its localization to mediate K48-linked polyubiquitination, subsequently contributing to the modulation of the expression of JAK-STAT signaling suppressors.IMPORTANCE The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. However, the involvement of NS1 in DNA methylation during IAV infection has not been established. Here, we reveal that the NS1 protein binds the cellular DNMT3B DNA methyltransferase, thereby inhibiting the methylation of the promoters of genes encoding suppressors of JAK-STAT signaling. As a result, these suppressor genes are induced, and JAK-STAT signaling is inhibited. Furthermore, we demonstrate that the NS1 protein transports DNMT3B to the cytoplasm for ubiquitination and degradation. Thus, we identify the NS1 protein as a potential trigger of the epigenetic deregulation of JAK-STAT signaling suppressors and illustrate a novel mechanism underlying the regulation of host immunity during IAV infection.

ANDC: an early warning score to predict mortality risk for patients with Coronavirus Disease 2019.

BACKGROUND: Patients with severe Coronavirus Disease 2019 (COVID-19) will progress rapidly to acute respiratory failure or death. We aimed to develop a quantitative tool for early predicting mortality risk of patients with COVID-19. METHODS: 301 patients with confirmed COVID-19 admitted to Main District and Tumor Center of the Union Hospital of Huazhong University of Science and Technology (Wuhan, China) between January 1, 2020 to February 15, 2020 were enrolled in this retrospective two-centers study. Data on patient demographic characteristics, laboratory findings and clinical outcomes was analyzed. A nomogram was constructed to predict the death probability of COVID-19 patients. RESULTS: Age, neutrophil-to-lymphocyte ratio, D-dimer and C-reactive protein obtained on admission were identified as predictors of mortality for COVID-19 patients by LASSO. The nomogram demonstrated good calibration and discrimination with the area under the curve (AUC) of 0.921 and 0.975 for the derivation and validation cohort, respectively. An integrated score (named ANDC) with its corresponding death probability was derived. Using ANDC cut-off values of 59 and 101, COVID-19 patients were classified into three subgroups. The death probability of low risk group (ANDC < 59) was less than 5%, moderate risk group (59 ≤ ANDC ≤ 101) was 5% to 50%, and high risk group (ANDC > 101) was more than 50%, respectively. CONCLUSION: The prognostic nomogram exhibited good discrimination power in early identification of COVID-19 patients with high mortality risk, and ANDC score may help physicians to optimize patient stratification management.