Given the significant clinical and economic burden of metabolic dysfunction-associated steatohepatitis (MASH), there is an urgent imperative to develop effective preventive and therapeutic agents. In this study, we evaluated the preventive effects and potential mechanisms of plant-derived compounds inositols (INS) and inositol hexakisphosphate (IP6) against MASH, utilizing a mouse model induced by western diet combined with low doses of CCl4 (WD/CCl4). The results showed that pretreatment with IP6 or INS significantly reduced obesity, fibrosis and lipid accumulation, alleviated insulin resistance, and prevented hyperglycemia in MASH mice. Both IP6 and INS also alleviated free fatty acid (FFA)- induced lipid accumulation in HepG2 cells. Transcriptomics analysis of the liver revealed that IP6 and INS reversed MASH-related transcriptomic changes, suppressing the expression of pro-fibrotic, pro-inflammatory, and lipid-metabolic genes. Integrated metabolomics and lipidomics revealed that the preventive effect of IP6 and INS against MASH was mediated through the rectification of disordered glycerophospholipid metabolism. Notably, knockdown of PEMT expression abolished the inhibitory effects of IP6 and INS on lipid accumulation, as well as α-SMA and COL-1 expression, in in vitro assays. These findings provide evidence that both IP6 and INS can prevent the development of MASH by improving disorders in glycerophospholipid metabolism, at least partially through the upregulation of PEMT.

SARS-CoV-2 infection remains a global health threat, yet its pathogenic mechanisms are incompletely understood. Here, we show that viral nucleocapsid protein (NP) is detectable in the serum of patients with SARS-CoV-2 infection independently of viral RNA. Using virus-like particle (VLP) systems and in vitro infection models, we demonstrate that NP is secreted in a vesicle-free form via type I unconventional protein secretion (UPS) pathway. This process is regulated by NP phosphorylation and oligomerization, coordinated by viral structural proteins, and dependent on membrane components including heparan sulfate proteoglycans (HSPGs) and phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2). Secreted NP is preferentially taken up by granulocytes and induces the release of inflammatory cytokines, including IL-6 and TNF-α. Our findings shed a light on the secretion mechanism of this pro-inflammatory NP, highlighting it as a potential therapeutic target for COVID-related systemic inflammation.

The underlying mechanisms of incomplete response to ursodeoxycholic acid (UDCA) therapy in patients with primary biliary cholangitis (PBC) remain unclear. This study aimed to investigate the clinicopathological characteristics and potential mechanisms of hepatobiliary (HB) cells in PBC patients with an incomplete response to UDCA. A total of 132 PBC patients who underwent ultrasound-guided liver biopsy were enrolled. Liver tissue samples were processed using multiple histochemical staining methods. Demographic, clinical, hematological, autoantibody, and biochemical data were retrospectively analyzed. The therapeutic response to UDCA was evaluated according to the Paris II criteria. Additionally, bile acid metabolomic profiling was performed using liquid chromatography-mass spectrometry (LC-MS) on paraffin-embedded liver tissues from 25 randomly selected PBC patients. Among the 132 patients, 58 (43.9

IntroductionThe efficacy of oncolytic adenoviruses in colorectal cancer models is constrained by a treatment-induced limitation: the high-dose, repetitive administration required for sustained oncolysis promotes chronic antigen exposure and tumor microenvironmental stress, driving CD8+ T cells into a state of exhaustion.MethodsTo mitigate this, we constructed an oncolytic adenovirus, ADV-PTD4-D3, engineered for intratumoral expression of a peptide inhibitor of CDK4/6. This local strategy aims to retain immunomodulatory potential while minimizing systemic exposure. In syngeneic murine models, ADV-PTD4-D3 demonstrated improved tumor control and the ability to induce robust, antigen-specific immunological memory, with its therapeutic effect being primarily dependent on CD8+ T cells. Notably, it also exhibited potent antitumor activity in a humanized xenograft model and showed no evidence of significant off-target toxicity in immunocompetent hosts.ResultsThe mechanism involves a signaling axis where viral-mediated CDK4/6 inhibition reduces retinoblastoma (Rb) protein phosphorylation. This decrease relieves Rb-mediated sequestration of the NF-kB p65 subunit, allowing p65 nuclear translocation and transcriptional upregulation of the T-cell chemoattractant CCL5, a factor linked to favorable patient prognosis. Thus, ADV-PTD4-D3 promotes a T-cell-inflamed microenvironment by providing a sustained chemotactic signal CCL5 for CD8+ T cell recruitment. Furthermore, this treatment strategy successfully reverses the functional exhaustion of infiltrating CD8+ T cells, thereby addressing two major barriers to effective therapy: inadequate infiltration and functional exhaustion. By modifying the tumor microenvironment in this way, the armed virus addresses two factors that limit T-cell-based immunotherapies: inadequate infiltration and functional exhaustion. Correspondingly, ADV-PTD4-D3 treatment improved the antitumor response to both PD-1 blockade and CAR-T cell therapy in combination studies.DiscussionThese findings suggest that engineering oncolytic viruses to locally modulate pathways involved in T cell exhaustion represents a viable and translatable strategy for enhancing antitumor immunity.

Elucidation of the regulation mechanism of hepatitis B virus (HBV) replication will provide potential targets for the development of novel anti-HBV therapeutics. It has been reported that the N6-methyladenosine (m6A) modification of HBV RNA plays a crucial role in the HBV life cycle. However, the mechanisms underlying the regulation of this modification remain incompletely understood. In this study, combining loss- and gain-of-function genetic analyses, we defined the role of IGF2BP1, an m6A reader, in facilitating HBV replication. Mechanistic studies revealed that IGF2BP1 stabilizes HBV RNAs primarily by binding to m6A-modified A1907 sites through its KH3-4 domain, thereby enhancing viral replication. Furthermore, targeted inhibition of IGF2BP1 by Cucurbitacin B, a small molecule inhibitor of IGF2BP1, was shown to inhibit HBV replication in vitro and in vivo. Taken together, these findings identify IGF2BP1 as a critical host regulator of HBV RNA stability through an m6A-dependent manner and targeted inhibition of IGF2BP1 effectively attenuates viral replication, providing a promising strategy for anti-HBV drug development.

BACKGROUND & AIMS:A notable proportion of CHB patients undergoing PEG-IFNα based therapy experience lagged serum HBeAg and/or HBV DNA disappearance in patients achieving HBsAg loss. In this study, we explored the molecular mechanisms behind this clinical phenomenon, offering novel insights into the sustainability of chronic HBV infection. METHODS:Two independent clinical cohorts were enrolled to validate this phenomenon. Then comprehensive analysis was performed using public datasets, coupled with a series of molecular biology experiments. RESULTS:Approximately 17-20% CHB patients underwent PEG-IFNα based therapy experienced seroclearance of HBsAg, while serum HBeAg and/or HBV DNA remained positive. These patients are more prone to serum HBsAg reappearance compared to those achieving complete virological response. Analysis of public datasets revealed that compared to the PC/BCP, the SP1/SP2 promoter displayed more pronounced inhibitory epigenetic modifications in HBeAg-negative patients and SP1/SP2 in-frame mutation peaked in immune active patients. In vitro experiments demonstrated that introduced SP1/SP2 inactive mutations would enhance PC/BCP transcriptional activity by a mechanism known as adjacent transcriptional interference. Furthermore, the deletion of L-HBsAg facilitated intracellular cccDNA replenishment. CONCLUSION:This study elucidates that under IFNα treatment and low viral load, transcriptional suppression of SP1/SP2 promoters through mutations and/or epigenetic changes would favour the maintenance of sustain chronic HBV infection, via enhancing the transcription activity of BCP to promote cccDNA replenishment. IMPACT AND IMPLICATIONS:In clinical practice with IFNα antiviral treatment for CHB patients, a "paradoxical" phenomenon is observed where serum HBsAg disappears while HBV DNA or/and HBeAg remains at low positive levels, with delayed disappearance. Our study confirms this clinical phenomenon using two independent clinical cohorts and explores the potential mechanisms behind the persistence of chronic HBV infection under IFNα treatment and low viral load. Transcriptional suppression of SP1/SP2 promoters through mutations and/or epigenetic changes supports the maintenance of chronic HBV infection by enhancing the transcriptional activity of the BCP, which in turn promotes cccDNA replenishment. HighlightsApproximately 20% of patients with CHB who have just achieved HBsAg loss under IFNα treatment show positive serum HBV DNA and/or HBeAg.During disease progression, in frame indel mutations accumulate in the HBV genome's SP1 and SP2 promoters, with epigenetic modifications contributing to their suppression.In frame indel mutations in the HBV genome's SP1 and SP2 promoters inhibit the transcription of HBV S mRNA and promote the transcription of 3.5 kb HBV RNA.The loss of L-HBs and envelop proteins leads to an increase in intracellular cccDNA, promoting the maintenance of chronic infection.

Background:Mycobacterial infections represent a major cause of morbidity and mortality in HIV-infected individuals. This study evaluated diagnostic techniques for mycobacterial identification and compared clinicopathological features between HIV-positive and HIV-negative patients. Methods:We analyzed 88 tissue samples (with 41 matched blood and 28 sputum samples) using histopathology (HE and acid-fast staining), bacterial culture, MTB-PCR (sputum/biopsy), PCR-reverse dot blot hybridization (RDBH), and metagenomic pathogen detection technology (MetaPath™). Logistic regression analyses were performed to identify factors affecting detection rates. Results:Mycobacterial infection was detected in 95.5% (84/88) of patients. Among HIV-positive patients (n=63), 46% (29/63) had Mycobacterium tuberculosis (MTB) infections, and 44% (28/63) had non-tuberculous mycobacteria (NTM) infections, significantly higher than the 20% (5/25) NTM rate in HIV-negative patients. Univariate analysis identified HIV-positive status (p=0.009), lymph node involvement (p=0.020), and positive MetaPath™ results (p=0.002) as significant predictors of detection, while multivariate analysis confirmed these as independent factors (p=0.036; p=0.042; p=0.006). Lymph nodes were the most common infection site in HIV-positive patients (42.9%, 27/63), while lung tissue predominated in HIV-negative patients (48%, 12/25). MetaPath™ demonstrated superior sensitivity and specificity for detecting both MTB and NTM. Biopsy samples provided higher diagnostic accuracy than sputum or blood for lung and lymph node infections, but not for brain. In HIV-positive patients, NTM infections showed significantly more granuloma formation (p=0.032) and foam cells (p=0.005), but less necrosis (p=0.0005) compared to MTB infections. No significant differences were observed in HIV-negative patients. Conclusions:MetaPath™ is a highly effective diagnostic tool for mycobacterial infections, particularly in tissue biopsies. HIV-positive status, lymph node involvement, and MetaPath™ positivity independently predict mycobacterial detection. HIV-positive patients exhibit distinct clinicopathological features, emphasizing the need for tailored diagnostic and therapeutic approaches based on immune status.

The persistence of covalently closed circular DNA (cccDNA) in hepatitis B virus (HBV)-infected hepatocytes remains a major obstacle to effective antiviral treatment. Understanding the molecular mechanisms regulating HBV cccDNA transcription is essential for developing novel therapeutic strategies. In this study, we investigated the role of RNA binding motif protein 25 (RBM25) in HBV replication, focusing on its interaction with cccDNA and its regulation of host transcription factors. The results demonstrated that RBM25 knockdown markedly inhibited HBV replication, reducing levels of HBV DNA, hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg), HBV RNA, and L-HBs in HBV-replicating and infected cell models. Consistent results were observed in a mouse model hydrodynamically injected with 1.2 ​× ​HBV plasmid. Conversely, RBM25 overexpression significantly enhanced HBV replication. Mechanistically, RBM25 promoted HBV promoter activities by binding to cccDNA through its RE/RD and PWI domains. This effect was mediated by increased Yin Yang 1 (YY1) expression, which enhanced acetylation of cccDNA-bound histones, promoting HBV transcription. Furthermore, RBM25 expression was upregulated and translocated to the nucleus following core protein expression and accumulation, while overexpression of RBM25 promoted core protein degradation. In conclusion, this study demonstrates that RBM25 is a novel host factor that enhances HBV replication by upregulating YY1-dependent transcriptional activation of cccDNA. It also reveales a reciprocal regulatory mechanism between the HBV core protein and RBM25, which helps sustain HBV replication.

The tumor immune microenvironment (TIME) played a crucial role in head and neck squamous cell carcinoma (HNSCC), significantly impacting disease progression and immunotherapy. To address these complexities, we analyzed gene expression profiles of 790 HNSCC tumors by combining data from The Cancer Genome Atlas (TCGA; n = 520) and the Gene Expression Omnibu database (GEO, n = 270). We identified five distinct TIME-based phenotypes, with the E class-characterized by tertiary lymphoid structures (TLS)-showing the highest survival rates and immunotherapy responses. To assess TLS prognostic value, we evaluated two cohorts of human papillomavirus (HPV) infection negative HNSCC: 247 surgical resection patients and 35 immunotherapy-treated patients with postoperative recurrence/metastasis. Additionally, we analyzed the GEO dataset (n = 102) comprising HNSCC patients with metastasis/recurrence who received PD-1/PD-L1 inhibitors, integrating clinical and transcriptomic data. Immune stratification systems integrating TIME composition and immune cell counts revealed that TLS-positive patients exhibited superior survival and enhanced immunotherapy responses in HPV-negative HNSCC. The two immune stratification systems exhibited greater accuracy and predictive power in estimating the overall survival of HPV-negative HNSCC patients compared to the use of TNM staging and tumor pathological stage. These findings provided a framework for establishing TLS-based personalized treatment in HPV-negative HNSCC, potentially enhancing disease surveillance, informing clinical practice, and improving patient prognosis.

Hepatitis B virus (HBV) infection remains a major public health problem, causing nearly one million deaths annually. Nucleoporin 153 (NUP153) is known to facilitate the nuclear entry of the human immunodeficiency virus (HIV) nucleocapsids, and recent studies suggest it also plays a role in HBV nucleocapsids nuclear import. We aimed to investigate the impact of NUP153 on HBV replication and its underlying mechanism. NUP153 was knocked down by RNA interference or CRISPR/Cas9-mediated gene disruption, or overexpressed using an expression plasmid in HBV-replicating cells and animal model. Luciferase reporter assays were employed to assess the activities of viral or host factor promoters. Cytoplasmic and nuclear fractionation experiments were conducted to analyze the subcellular distribution of proteins and HBV RNA. In the present study, we found that knockdown of NUP153 significantly inhibited HBV replication without affecting the levels of covalently closed circular DNA (cccDNA) in both the prcccDNA/Cre recombinant plasmid system and HepG2-NTCP cells. Consistent results were observed in a mouse model hydrodynamically injected (HDI) with 1.2 × HBV plasmid. Conversely, NUP153 overexpression markedly increased cccDNA transcription and progeny virus production. Further study revealed that NUP153 enhanced HBV core promoter activity, likely through a hepatocyte nuclear factor 4α (HNF4α)-dependent mechanism. Mechanistically, ERK signaling was essential for NUP153-mediated promotion of HNF4α and HBV replication. Additionally, HBV replication significantly upregulated NUP153 mRNA and protein levels in both HBV cell models and HBV-infected patients. Together, we identify NUP153 as a novel host factor that promotes HBV replication by enhancing cccDNA transcription through the upregulation of HNF4α, suggesting a potential therapeutic strategy for HBV replication.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Despite the proven efficacy of immunotherapy against malignancies, a large proportion of patients with HCC fail to benefit from these efficacious agents because of their overwhelmingly immunosuppressive microenvironment. Therefore, there is an urgent need to identify key genes and develop effective strategies for reshaping the HCC microenvironment. Here, a significant downregulation of C-X-C motif chemokine ligand 2 (CXCL2) in HCC is identified due to gene copy number loss, which correlates with poor prognosis and suboptimal responsiveness to immunotherapy. Subsequently, it is found that CXCL2 can not only recruit neutrophils as expected, but also induce their polarization toward the antitumor type to curb HCC progression. Mechanistically, differing from the prevailing notion that CXCL2 primarily functions extracellularly as a chemokine, it is demonstrated that intracellular CXCL2 can bind to Y-Box Binding Protein 1 (YBX1) and prevent its nuclear translocation. Consequently, this reduces the transcription of sterol regulatory element binding transcription factor 2 (SREBF2) and suppresses cholesterol biosynthesis, thereby remodeling HCC microenvironment and impeding HCC development. In summary, this study highlights the unconventional role of CXCL2 in regulating neutrophil polarization and immune responses in HCC, positioning it as a potential therapeutic target for HCC.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the transcriptional template in HBV replication, is transcriptionally regulated by multiple host proteins such as epigenetic factors and transcription factors. This study aims to identify novel host proteins interacting with cccDNA and regulating its activity in HBV replication. Mass spectrometry analysis identified 129 host proteins associated with biotinylated cccDNA surrogate HBVcircle. A siRNA library screening demonstrated that knockdown of DNAJC9, CEBPZ, and EIF3A in HepG2 cells transfected with HBVcircle reduced the levels of HBsAg and HBeAg in the supernatant. Knockdown of DNAJC9 in HBV replication and infection cell models restricted viral replication, while the DNAJC9 overexpression showed an opposite trend. DNA pull-down, cccDNA ChIP, and immunofluorescence experiments indicated that DNAJC9 can bind to cccDNA in a manner independent of histones and specific DNA sequences. Dual luciferase reporter assay demonstrated that knockdown of DNAJC9 reduces the transcriptional activity of HBV promoters and enhancers. Co-IP and cccDNA ChIP experiments showed that DNAJC9 can interact with histone H3.3, and knockdown of DNAJC9 reduced H3.3, H3K4me3, and H3K27ac on cccDNA. In the HepAD38 or HepG2-NTCP cells, HBV replication led to a decrease in the cytoplasmic distribution and an increase in the nuclear distribution of DNAJC9. Histone chaperone DNAJC9 can bind to cccDNA in a histone-independent manner. DNAJC9 upregulates cccDNA transcription and viral replication by increasing the density of H3.3, H3K4me3, and H3K27ac on cccDNA, thereby activating its promoters and enhancers. HBV replication may promote the nuclear localization of DNAJC9 protein, thus facilitating active transcription and replication of HBV.

Elucidating the molecular mechanisms underlying hepatitis B virus (HBV)-host interactions will hold promise for identifying novel therapeutic targets for HBV. This study aims to investigate the functional interplay and regulatory mechanisms between HBV and the AU-rich element RNA-binding protein 1 (AUF1). We demonstrate that AUF1 inhibits HBV replication and expression in HBV-infected HepG2-NTCP cells, HepG2 cells cotransfected with prcccDNA and pCMV-Cre plasmids, and HepAD38 cells. Mechanistically, AUF1 reduces HBV RNA levels post-transcriptionally without altering the transcriptional levels. This suppression occurs primarily through accelerated RNA decay, while translation efficiency remains unaffected. RNA immunoprecipitation confirms AUF1 associates with HBV RNA at multiple binding sites. However, AUF1 and human antigen R (HuR) neither compete for HBV RNA binding nor exhibit antagonistic regulatory functions. Intriguingly, HBV replication significantly reduces intracellular AUF1 protein levels without affecting AUF1 mRNA, suggesting viral induction of AUF1 proteolytic degradation. AUF1 acts as a novel posttranscriptional suppressor of HBV replication via RNA destabilization, revealing a dynamically regulated host-virus conflict where HBV counteracts AUF1 through protein degradation.

Hepatitis B virus (HBV) is the primary etiological agent of chronic hepatitis B (CHB) infection, posing a serious threat to human health. The pregenomic RNA (pgRNA) of HBV is the template for HBV reverse transcription, and the epsilon stem-loop (ε) is required for nucleocapsid assembly. The host factor serine/arginine (SR)-rich splicing factor 7 (SRSF7) is a splicing regulator and RNA-binding protein that was involved in regulating viral RNA splicing and export from the nucleus during the viral life cycle, but its biological function and regulatory mechanisms in HBV remain unclear. In this study, SRSF7 was found to promote HBV replication and upregulate HBV RNA levels through knockdown or overexpression of SRSF7 in different cell lines using the HBV replication model. Surprisingly, we found that SRSF7 enhanced HBV RNA stability at the post-transcriptional level, rather than regulating its splicing. We further demonstrated that SRSF7 could bind to pgRNA; deletion of the bulge and loop structures of the ε element significantly reduced its binding capacity. In addition, we confirmed that SRSF7 supports HBV replication in CHB patients. Our study suggests that the host factor SRSF7 promotes HBV replication, which provides new perspectives for further elucidation of HBV-host interactions and the development of host-targeted anti-HBV drugs.

The natural progression of chronic hepatitis B virus (HBV) infection is dynamic, but the longitudinal landscape of HBV serological markers with host antiviral immune response relevant hepatic inflammatory damage remains undetermined. To this issue, we studied the association of HBV serological markers with the severity of hepatic inflammatory damage and enumerated HBV-specific T cells using the cultured enzyme-linked immune absorbent spot (ELISpot). Five hundred and twenty-four treatment-naïve chronic HBV infection patients were enrolled. The Spearman correlation analysis revealed that in hepatitis B e antigen (HBeAg)-positive patients, all HBV virologic indicators negatively correlated with liver inflammatory damage and fibrosis (p < 0.01). Stronger correlations were accessed in the subgroup of HBeAg-positive patients with HBV DNA > 2 × 106 IU/mL (p < 0.01), whereas negative correlations disappeared in patients with HBV DNA ≤ 2 × 106 IU/mL. Surprisingly, in HBeAg-negative patients, the HBV DNA level was positively correlated with the hepatic inflammatory damage (p < 0.01). The relationship between type Ⅱ interferon genes expression and HBV DNA levels also revealed a direct shift from the initial negative to positive in HBeAg-positive patients with HBV DNA declined below 2 × 106 IU/mL. The number of HBV-specific T cells were identified by interferon γ ELISpot assays and showed a significant increase from HBeAg-positive to HBeAg-negative group. The host's anti-HBV immunity remains effective in HBeAg-positive patients with HBV DNA levels exceeding 2 × 106 IU/mL, as it efficiently eliminates infected hepatocytes and inhibits HBV replication. However, albeit the increasing number of HBV-specific T cells, the host antiviral immune response shifts towards dysfunctional when the HBV DNA load drops below this threshold, which causes more pathological damage and disease progression.

Objective: To describe the ultrasound characteristics of nodular localized cutaneous neurofibroma (NLCN).Materials and Methods; Clinical features and ultrasound characteristics of 43 lesions of 40 patients pathologically proven as NLCNs at Peking University Shenzhen Hospital from October 2014 to May 2022 were analyzed retrospectively. The location, length-to-thickness (L/T) ratio, thickness-to-width (T/W) ratio, shape, margin, capsule, echogenicity, echotexture, posterior features, vascularity, and "rat tail sign" were evaluated.Results: All ultrasound findings showed almost perfect agreement. More than a half of NLCNs (n = 24, 55.8%, p < 0.001) were located in the subcutaneous fat layer wholly with well-demarcation from dermis and deep fascia. Most of the NLCNs were fusiform shape (n = 27, 62.8%, p < 0.001) in the long axis and oval shape (n = 35, 81.4%, p < 0.001) in the short axis. The other ultrasound findings of NLCNs included well-defined (n = 42, 97.7%, p < 0.001), encapsulated (n = 39, 90.7%, p < 0.001), predominately hypoechoic (n = 34, 79.1%, p < 0.001), homogeneous (n = 39, 90.7%, p < 0.001), posterior enhancement (n = 29, 67.4%, p = 0.033), and avascularity (n = 37, 86.0%, p < 0.001). Only a quarter (n = 11, 25.6%, p = 0.002) of lesions were recognized with the "rat tail sign."Conclusion: NLCNs present as fusiform shape in long axis and round shape in short axis. The common ultrasound findings of NLCNs are well-defined, encapsulated, predominately hypoechoic, homogeneous lesion with posterior enhancement, and poor blood supply. The "rat tail sign" has low sensitivity in NLCNs.

Naturally occurred precore (PC, G1896A) and/or basal core promoter (BCP, A1762T/G1764A) mutations are prevalent in chronic HBV-infected patients, especially those under HBeAg-negative status. However, the replicative capacity of HBV with PC/BCP mutations remains ambiguous. Herein, meta-analysis showed that, only under HBeAg-negative status, the serum HBV DNA load in patients with PC mutation was 7.41-fold higher than those without the mutation. Both PC mutation alone and BCP+PC mutations promoted HBV replication in cell and hydrodynamic injection mouse models. In human hepatocyte chimeric mouse model, BCP+PC mutations led to elevated replicative capacity and intrahepatic core protein accumulation. Mechanistically, preC RNA harboring PC mutation could serve as mRNA to express core and P proteins, and such pgRNA-like function favored the maintenance of cccDNA pool under HBeAg-negative status. Additionally, BCP+PC mutations induced more extensive and severe human hepatocyte damage as well as activated endoplasmic reticulum stress and TNF signaling pathway in livers of chimeric mice. This study indicates that HBeAg-negative patients should be monitored on HBV mutations regularly and are expected to receive early antiviral treatment to prevent disease progression.