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Objective: Baicalin, which is a key bioactive constituent obtained from Scutellaria baicalensis, has been utilized in traditional Chinese medicine for many centuries. Although it has been reported that Baicalin (BA) can inhibit the replication of the Hepatitis B virus (HBV), the exact mechanism behind this process remains unclear. Interferon-stimulated genes (ISGs) are crucial in the process of antiviral defense. We aim to investigate whether BA can regulate the expression of ISGs, and thereby potentially modulate the replication of HBV. Methods: The study involved the use of CRISPR/Cas9 technology to perform knockout experiments on TRIM25 and IFIT3 genes. The expression of these genes was confirmed through techniques such as immunoblotting or Q-PCR. The levels of HBsAg and HBeAg were measured using ELISA, and the expression of interferon-stimulated genes was detected using a luciferase assay. Results: It is interesting to note that several ISGs belonging to the TRIM family, including TRIM5, TRIM25, and TRIM14, were induced after BA treatment. On the other hand, members of the IFIT family were reduced by BA stimulation. Additionally, BA-mediated HBV inhibition was found to be significantly restored in HepG2 cells where TRIM25 was knocked out. Additional research into the mechanism of action of BA found that prolonged treatment with BA activated the JAK/STAT signaling pathway while simultaneously inhibiting the NF-kB pathway. Conclusion: The findings of our study indicate that TRIM25 has a significant impact on the regulation of HBV replication following BA treatment, providing additional insight into the mechanisms by which BA exerts its antiviral effects.

Abstract. Background:. The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25. Methods:. The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay. Results:. We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process. Conclusions:. The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.

Hepatitis B, a global health concern caused by the hepatitis B virus (HBV), infects nearly 2 billion individuals worldwide, as reported by the World Health Organization (WHO). HBV, a hepatotropic DNA virus, predominantly targets and replicates within hepatocytes. Those carrying the virus are at increased risk of liver cirrhosis and hepatocellular carcinoma, resulting in nearly 900,000 fatalities annually. The HBV X protein (HBx), encoded by the virus’s open reading frame x, plays a key role in its virulence. This protein is integral to viral replication, immune modulation, and liver cancer progression. Despite its significance, the precise molecular mechanisms underlying HBx remain elusive. This review investigates the HBx protein’s roles in HBV replication, interferon signaling regulation, and hepatocellular carcinoma progression. By understanding the complex interactions between the virus and its host mediated by HBx, we aim to establish a solid foundation for future research and the development of HBx-targeted therapeutics.

Abstract Background Ulcerative colitis (UC) is a form of chronic inflammatory bowel disease of nonspecific origin. This study used an RNA-Sequencing (RNA-Seq) approach to evaluate the transcriptomic landscape of a well-stratified treatment-naïve pediatric UC patient population by comparing them with healthy control children. The data were analyzed to evaluate the mechanisms driving UC-related intestinal inflammation and fibrosis. Methods Intestinal mucosal samples from five pediatric UC patients and five healthy controls were analyzed by RNA-Seq, and results were verified by qPCR. A CRISPR/Cas9 approach was used to knock out the expression of HLA-DRB5, and molecular biology techniques were used for additional mechanistic studies. Results In these analyses, 2290 genes were found to be differentially expressed between the UC and control samples, of which 1258 and 1032 were upregulated and downregulated, respectively. Gene Ontology analysis showed that these genes were enriched in extracellular matrix (ECM)-related processes and that 7 of 8 differentially expressed genes of interest (PIK3CD, IL1β, IL1α, TIMP1, MMP1, MMP12, COL6A3, and HLADRB5) were upregulated and involved in ECM-receptor interaction and inflammatory bowel disease-related pathways. Increased HLA-DRB5 expression driven by intestinal bacteria was found to promote IL-1α secretion, leading to intestinal inflammation and fibrosis, suggesting a possible target for the treatment of UC. Conclusion These data suggest that intestinal inflammation is present in pediatric UC patients for extended periods before the onset of symptoms, and intestinal fibrosis begins even during the early stages of UC. Intestinal bacteria were also found to trigger intestinal inflammation and fibrosis, with HLA-DRB5 playing a central role in this process.

Abstract The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and overactive immune response in the late stage of infection; However, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein dually regulated the phosphorylation and nuclear translocation of IRF3, STAT1, and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress the ubiquitination and activation of retinoic acid-inducible gene I (RIG-I). Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.

Hepatitis B virus (HBV) infection in humans and its associated diseases are long-standing problems. HBV can produce a large number of non-self-molecules during its life cycle, which acts as targets for innate immune recognition and initiation. Among these, interferon and its large number of downstream interferon-stimulated gene molecules are important early antiviral factors. However, the development of an effective antiviral immune response is not simple and depends not only on the delicate regulation of the immune response but also on the various mechanisms of virus-related immune escape and immune tolerance. Therefore, despite there being a relatively well-established consensus on the major pathways of the antiviral response and their component molecules, the complete clearance of HBV remains a challenge in both basic and clinical research. Long-noncoding RNAs (lncRNAs) are generally >200 bp in length and perform different functions in the RNA strand encoding the protein. As an important part of the IFN-inducible genes, interferon-stimulated lncRNAs are involved in the regulation of several HBV infection-related pathways. This review traces the basic elements of such pathways and characterizes the various recent targets of lncRNAs, which not only complement the regulatory mechanisms of pathways related to chronic HBV infection, fibrosis, and cancer promotion but also present with new potential therapeutic targets for controlling HBV infection and the malignant transformation of hepatocytes.

ABSTRACT Hepatitis B virus (HBV)‐related diseases are among the major diseases that affect millions of people worldwide. These diseases are difficult to eradicate and thus pose a serious global health challenge. There is an urgent need to understand the cross talk mechanism between HBV and the host. Cholesterol‐25‐hydroxylase (CH25H) and its enzymatic product, 25‐hydroxycholesterol (25HC), were previously shown to exhibit effective broad‐spectrum antiviral activity. However, the role of CH25H in the regulation of HBV infection and replication remains unclear. The present study reported increased expression of CH25H in HBV-infected patients compared to healthy subjects. Importantly, higher expression of CH25H expression was found to be associated with low HBV replication. Additionally, the present study aimed to identify CH25H mutants, which would lack hydroxylase activity but retain antiviral activity toward HBV infection and replication. Interestingly, it was observed that both CH25H and its mutants interacted with HBx protein and inhibited nuclear translocation of HBx. In particular, CH25H interacted with the C-terminal region of HBx, while transmembrane region 3 of CH25H was found to be critical for CH25H–HBx interaction and inhibition of HBV replication. The study results suggested that 25HC promoted HBV infection but not HBV replication. Thus, the results of the present study suggested the involvement of a dual mechanism in CH25H-mediated regulation of HBV replication. The study clearly demonstrated cross talk between HBV and the host through CH25H–HBx axis. IMPORTANCE The enzymatic product of CH25H, 25-hydroxycholesterol (25HC), has been previously shown to play a critical role in the blockage of the cell-virus fusion in response to viral infection. However, our study indicates a dual role of CH25H in regulating HBV. We find the CH25H-mediated inhibition of HBV replication is independent on its enzyme activity and CH25H binds to HBx and inhibits HBx nucleus translocation. We are interested to find out 25HC promotes HBV infection.

Owing to its broad-spectrum antivirus activities, interferon (IFN) is an important alternative agent for use in the treatment of hepatitis B virus (HBV)-infected patients; however, the mechanism involved in the inhibition of HBV infection and replication by IFN remains unclear. We previously reported that the induction of TRIM5γ is important in the IFN treatment of HBV patients as it promotes the degradation of the HBx protein, while the manner in which TRIM5γ is induced by IFN and how TRIM5γ interacts with HBx remain unestablished until date. Our present findings confirmed the TRIM5γ-HBx-DDB1 interactions in the HBV-infected Primary human hepatocytes (PHH), and we further found that STAT3, and not STAT1, was responsible for the induction of TRIM5γ upon IFN stimulation and that the zinc binding site His123 on the BBOX domain was a decisive site in the interaction between TRIM5γ BBOX and HBx. In addition, based on the BBOX domain, we detected a 7-amino acid peptide with the potential of promoting HBx degradation and inhibiting HBV replication. On the other hand, we noted that the TRIM5γ expression was inhibited by HBV in chronically HBV infected patients. Thus, our study identified the crucial role of STAT3 in the induction of TRIM5γ, as well as proposed a 7-amino acid, small peptide as a potential candidate for the development of therapeutic agents targeting HBx.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of current COVID-19 pandemic, and insufficient production of type I interferon (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS-CoV-2 has been reported to suppress host IFN-I production, but the underlying mechanism is not completely understood. In this study, SARS-CoV-2 M protein was confirmed to suppress the expression of IFNβ and interferon-stimulated genes induced by RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further impaired the formation of TRAF3–TANK–TBK1-IKKε complex that leads to inhibition of IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative regulation of IFN-I production, which would provide deeper insight into the innate immunosuppression and pathogenicity of SARS-CoV-2.

Summary: To understand the molecular mechanisms that mediate the anti-hepatitis B virus (HBV) effect of interferon (IFN) therapy, we conduct high-throughput bimolecular fluorescence complementation screening to identify potential physical interactions between the HBx protein and 145 IFN-stimulated genes (ISGs). Seven HBx-interacting ISGs have consistent and significant inhibitory effects on HBV replication, among which TRIM5γ suppresses HBV replication by promoting K48-linked ubiquitination and degradation of the HBx protein on the K95 ubiquitin site. The B-Box domain of TRIM5γ under overexpression conditions is sufficient to trigger HBx degradation and is responsible both for interacting with HBx and recruiting TRIM31, which is an ubiquitin ligase that triggers HBx ubiquitination. High expression levels of TRIM5γ in IFN-α-treated HBV patients might indicate a better therapeutic effect. Thus, our studies identify a crucial role for TRIM5γ and TRIM31 in promoting HBx degradation, which may facilitate the development of therapeutic agents for the treatment of patients with IFN-resistant HBV infection. : In brief, Tan et al. find that IFN-induced TRIM5γ recruits TRIM31 to degrade HBx, resulting in suppression of hepatitis B virus replication. Keywords: type I IFN, HBV, HBx, TRIM5γ, TRIM31

Therapeutic administration of type I IFN (IFN-I) is a common treatment option for individuals suffering from hepatitis B virus (HBV) infection. IFN-I therapy, however, has a relatively low response rate in HBV-infected patients and can induce serious side-effects, limiting its clinical efficacy. There is, thus, a clear need to understand the molecular mechanisms governing the influence of IFN-I therapy in HBV treatment in order to improve patient outcomes. In this study, we explored the interactions between HBV and IFITs (IFN-induced proteins with tetratricopeptide repeats), which are classical IFN-inducible genes. Specifically, we found that HBV patients undergoing IFN-I therapy exhibited elevated expression of IFITs in their peripheral blood mononuclear cells (PBMCs). We further observed upregulation in the expressions of IFIT1, IFIT2, and IFIT3 in cells transfected with the pHBV1.3 plasmid, which yields infectious virions in hepatic cells. We additionally found that HBx, which is the only regulatory protein encoded within the HBV genome, activates NF-κB, which in turn directly drives IFIT3 transcription. When IFIT3 was overexpressed in HepG2 cells, HBV replication was enhanced. Together, these results suggest that IFIT genes may unexpectedly enhance viral replication, thus making these genes potential therapeutic targets in patients with HBV.

Dendritic cells instruct different types of T cell responses depending on the types of microbial ligands sensed. Here the authors show that dendritic cells stimulated via Dectin-1 receptor upregulate TNFSF15 and OX40L and induce T helper 9 response and efficient antitumour immunity in mouse models.

Hepatitis B virus (HBV) remains a major cause of hepatic disease that threatens human health worldwide. Type I IFN (IFN-I) therapy is an important therapeutic option for HBV patients. The antiviral effect of IFN is mainly mediated via upregulation of the expressions of the downstream IFN-stimulated genes. However, the mechanisms by which IFN induces ISG production and inhibits HBV replication are yet to be clarified. TRIM14 was recently reported as a key molecule in the IFN-signaling pathway that regulates IFN production in response to viral infection. In this study, we sought to understand the mechanisms by which IFN restricts HBV replication. We confirmed that TRIM14 is an ISG in the hepatic cells, and that the pattern-recognition receptor ligands polyI:C and polydAdT induce TRIM14 dependent on IFN-I production. In addition, IFN-I-activated STAT1 (but not STAT3) directly bound to the TRIM14 promoter and mediated the induction of TRIM14. Interestingly, TRIM14 played an important role in IFN-I-mediated inhibition of HBV, and the TRIM14 SPRY domain interacted with the C-terminal of HBx, which might block the role of HBx in facilitating HBV replication by inhibiting the formation of the Smc-HBx–DDB1 complex. Thus, our study clearly demonstrates that TRIM14 is a STAT1-dependent ISG, and that the IFN-I–TRIM14–HBx axis shows an alternative way to understand the mechanism by which IFN-I inhibits virus replication.

Chronic hepatitis B virus (HBV) infection imposes a severe burden on global public health. Currently, there are no curative therapies for millions of chronic HBV-infected patients (Lok et al., 2017). Interferon (IFN; including pegylated IFN) is an approved anti-HBV drug that not only exerts direct antiviral activity, but also augments immunity against HBV infection. Through a systematic review of the literature, here we summarize and present recent progress in research regarding the interactions between IFN and HBV as well as dissect the antiviral mechanisms of IFN. We focus on inhibition of HBV replication by IFN-stimulated genes (ISGs) as well as inhibition of IFN signaling by HBV and viral proteins. Finally, we briefly discuss current IFN-based HBV treatment strategies. This review may help to better understand the mechanisms involved in the therapeutic action of IFN as well as the crosstalk between IFN and HBV, and facilitate the development of both direct-acting and immunology-based new HBV drugs.

UV-induced stress response involves expression change of a myriad of genes, which play critical roles in modulating cell cycle arrest, DNA repair, and cell survival. Alteration of micro- RNAs has been found in cells exposed to UV, yet their function in UV stress response remains elusive. Here, we show that UV radiation induces up-regulation of miR-125b, which negatively regulates p38α expression through targeting its 3'-UTR. Increase of miR-125b depends on UV-induced NF-κB activation, which enhances miR-125b gene transcription upon UV radiation. The DNA damage-responsive kinase ATM (ataxia telangiectasia mutated) is indispensable for UV-induced NF-κB activation, which may regulate p38α activation and IKKβ-dependent IκBα degradation in response to UV. Consequently, repression of p38α by miR-125b prohibits prolonged hyper activation of p38α by UV radiation, which is required for protecting cells from UV-induced apoptosis. Altogether, our data support a critical role of NF-κB-dependent up-regulation of miR-125b, which forms a negative feedback loop to repress p38α activation and promote cell survival upon UV radiation [ABSTRACT FROM AUTHOR]