Xu Lin

Post-translational modifications orchestrate virus–host interactions, yet their roles in the hepatitis B virus (HBV) life cycle remain incompletely defined. The small hepatitis B surface antigen (SHBs)—the most abundant HBV protein—drives immune evasion, pathogenesis, and virion/subviral particle (SVP) production. Here, we identify UFL1, the E3 ligase of UFMylation, as a host factor that modifies and stabilizes SHBs by antagonizing its ubiquitination to potentiate HBV output and SHBs-driven pro-oncogenic activities. Using affinity purification-mass spectrometry, we discovered UFL1 as a SHBs interactor and validated the interaction by co-immunoprecipitation, confocal co-localization, and GST pull-down assays. In vivo and in vitro UFMylation assays demonstrated that UFL1 catalyzes SHBs’ UFMylation. Site-directed mutagenesis mapped the modification to K122; the K122R mutant failed to undergo UFMylation. Cycloheximide chase and ubiquitination assays showed that UFMylation at K122 stabilizes SHBs by antagonizing TRIM21-mediated K48-linked polyubiquitination and proteasomal degradation. Functionally, UFL1 enhanced SHBs-driven hepatocellular carcinoma cell migration and endothelial tube formation, whereas UFL1 knockdown produced opposite effects. In HBV-producing cells, UFL1 increased intracellular and extracellular HBsAg and HBV virions, with no effects on HBV RNA, indicating post-translational control of HBsAg and virion/SVP production. These findings establish SHBs as, to our knowledge, the first HBV protein substrate of UFMylation and reveal a UFL1-SHBs axis that promotes SHBs stability, pathogenic functions, and HBV particle output. Targeting UFMylation or the UFL1-SHBs interface offers a host-directed strategy to reduce HBsAg and virion production and may inform therapeutic approaches for HBV infection and HBV-related liver disease. IMPORTANCE: Chronic infection with hepatitis B virus (HBV) remains a major global health problem, in part because current treatments rarely reduce the viral surface protein that floods the blood and helps the virus persist. In this study, we asked how the most abundant viral surface protein—SHBs—is controlled inside cells. We discovered that UFL1 attaches a small “molecular tag” as UFM1 to this viral protein, and this tag protects the SHBs from the cell’s disposal system. As a result, the increased SHBs promotes behaviors linked to liver disease progression, and cells release more virion/SVPs. When we prevented this tagging event or changed a single amino acid on the SHBs so it could not be tagged, the protein became unstable and was degraded, and virus output dropped. Thus, to our knowledge, SHBs is the first identified HBV protein substrate of UFMylation, suggesting targeting the UFL1-SHBs interface may reduce HBsAg and virion production and inform HBV therapy.

Gastric cancer (GC) is frequently associated with lymph node metastasis, which contributes to a poor prognosis; however, the underlying molecular mechanisms remain incompletely understood. In this study, we identify the N6-methyladenosine (m⁶A) reader IGF2BP1 as a critical driver of lymphatic metastasis in GC. IGF2BP1 is significantly upregulated in GC tissues with lymph node metastasis and is correlated with adverse clinical outcomes. Mechanistically, IGF2BP1 binds to the m⁶A-modified 3′ untranslated region (3′UTR) of METTL21A mRNA, thereby enhancing its mRNA stability and expression levels. METTL21A, a non-histone lysine methyltransferase, interacts with and methylates the transcription factor SOX18, facilitating its nuclear translocation without altering protein stability. Once in the nucleus, SOX18 directly activates the transcription of CXCL12. Secreted CXCL12 promotes lymphangiogenesis and facilitates lymphatic metastasis through CXCR4 signaling. Functionally, both IGF2BP1 and METTL21A enhance tube formation and migration of lymphatic endothelial cells and promote lymphatic metastasis in GC. Notably, METTL21A knockout abolishes IGF2BP1-induced lymphangiogenesis, whereas SOX18 depletion eliminates the pro-lymphangiogenic effects of IGF2BP1. Our findings reveal a previously unrecognized signaling axis—IGF2BP1–METTL21A–SOX18–CXCL12—that links m⁶A-dependent post-transcriptional regulation with methylation-dependent transcriptional activation, offering potential therapeutic targets for the prevention of lymphatic metastasis in gastric cancer.

Hepatitis B virus (HBV) has been implicated in hepatocellular carcinoma (HCC) progression, partly through regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Although transcriptional regulation of PTEN by HBV is well characterized, its post-transcriptional regulation remains poorly understood. Because RNA 5-methylcytosine (m⁵C) modification influences post-transcriptional gene control and cancer development, we investigated whether HBV modulates PTEN through m⁵C. Methylated RNA immunoprecipitation (MeRIP)-quantitative polymerase chain reaction showed a marked reduction in m⁵C on PTEN mRNA in HBV-producing cells. MeRIP sequencing further identified decreased m⁵C within the PTEN coding sequence region (chr10:89717747-89717771) in HBV-producing HepAD38/tetracycline-off cells, with chr10:89717756 emerging as a critical site where HBV suppresses m⁵C enrichment and PTEN expression. Mechanistically, the m⁵C "writer" NOP2/Sun RNA methyltransferase 2 (NSUN2) and the "reader" Y-box binding protein 1 (YBX1) stabilized PTEN mRNA in an m⁵C-dependent manner. HBV disrupted this pathway, decreasing PTEN mRNA stability via NSUN2- and YBX1-mediated m⁵C. Overexpression of NSUN2 or YBX1 attenuated HBV-driven proliferation, migration, and invasion, and these effects were partially reversed by the PTEN inhibitor VO-Ohpic. The small hepatitis B surface antigen and hepatitis B X protein downregulated NSUN2 and YBX1, linking viral proteins to PTEN suppression. Further, HBV is associated with reduced NSUN2 expression in HBV transgenic (HBV-Tg) mice, HBV-infected primary human hepatocytes as well as HBV-positive clinical HCC specimens, supporting the physiological and clinical relevance of this finding. Together, these findings identify the NSUN2/YBX1/PTEN axis as a potential therapeutic target in HBV-associated HCC.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide, with a significant association to hepatitis B virus (HBV) infection, which has been shown to drive HCC progression. Sorafenib, a multi‐kinase inhibitor, is the first‐line treatment for advanced HCC. However, recent studies indicate that HBV infection may confer resistance to sorafenib treatment. The small hepatitis B surface antigen (SHBs), the most abundant HBV viral protein, has been implicated in HCC development, yet its role in sorafenib resistance is unclear. This study demonstrates that SHBs promotes sorafenib resistance in HCC cells and xenograft models by inhibiting apoptosis. Upon sorafenib treatment, SHBs expression was found to enhance the RAF1/MEK/ERK signaling pathway, as evidenced by increased phosphorylation of ERK and MEK. Inhibition of ERK activity with U0126 countered SHBs effects on sorafenib‐induced apoptosis, cleaved caspase‐3, and cellular proliferation. Mechanistically, SHBs binds to protein tyrosine phosphatase non‐receptor type 1 (PTPN1), enhancing its phosphorylation, which subsequently dephosphorylates the protein tyrosine phosphatase interacting protein 51 (PTPIP51). This dephosphorylation promotes RAF1 recruitment to the 14–3‐3β complex, leading to activation of the RAF1/MEK/ERK pathway. These findings suggest that SHBs prevents sorafenib‐induced apoptosis in HCC cells by binding to PTPN1 and stimulating the formation of the PTPIP51/14–3‐3β/RAF1 complex, thereby activating the RAF1/MEK/ERK signaling pathway. This mechanism provides insight into HBV‐induced sorafenib resistance in HCC, highlighting SHBs as a potential target for overcoming treatment resistance in HBV‐related HCC.

Epstein–Barr virus (EBV) is the first oncogenic DNA virus known to encode microRNAs (miRNAs) and has been implicated in the pathogenesis of multiple malignancies, including a distinct subset of gastric cancers (EBV-associated gastric cancer, EBVaGC). However, the functional roles of individual EBV-encoded miRNAs in EBVaGC remain poorly defined. In this study, we integrate bioinformatic and experimental analyses to uncover a novel oncogenic axis driven by EBV-encoded miR-BART20-3p. Analysis of public transcriptomic datasets revealed that peroxisome proliferator-activated receptor α (PPARα) is significantly downregulated in EBVaGC compared with EBV-negative gastric tumors. We confirmed that both PPARα mRNA and protein are reduced in EBVaGC cell lines and primary tumor specimens, and that this reduction inversely correlates with miR-BART20-3p levels. A dual-luciferase reporter assay demonstrated that miR-BART20-3p directly binds the PPARα 3′-UTR. Functionally, miR-BART20-3p overexpression in AGS cells enhanced proliferation and migration, whereas inhibition of miR-BART20-3p in EBV-infected AGS cells attenuated these phenotypes. Mechanistic studies employing PPARα-specific siRNA together with qRT-PCR and ELISA reveal that suppression of PPARα or overexpression of miR-BART20-3p leads to upregulation of interleukin 6 (IL-6), indicating disruption of the PPARα–IL-6 regulatory axis. Collectively, EBV-encoded miR-BART20-3p promotes EBVaGC progression by directly targeting PPARα, and thereby derepressing IL-6 expression. This miRNA–PPARα–IL-6 pathway may serve as both a mechanistic biomarker and a novel therapeutic target in EBVaGC.

The small hepatitis B surface antigen (SHBs) is the most abundant hepatitis B virus (HBV) protein in individuals infected with HBV, and clearance of HBV surface antigen, which is primarily composed of SHBs, is considered a surrogate biomarker for achieving a functional cure of chronic HBV. Understanding SHBs degradation is crucial for its elimination and targeted eradication strategies. This study demonstrates that SHBs undergoes degradation via a ubiquitin/proteasome pathway, primarily through K48-linked ubiquitination, with K122 as the critical ubiquitination site. Utilizing immunoprecipitation and mass spectrometry, we identified TRIM21 (an E3 ubiquitin ligase) and OTUD4 (a deubiquitinase) as key regulators of SHBs. We verified the direct interaction between SHBs and TRIM21’s coiled-coil domain, as well as the N-terminal amino acids 1–180 of OTUD4, using coimmunoprecipitation and glutathione S-transferase (GST) pull-down assays in both in vivo and in vitro settings. TRIM21 was observed to reduce SHBs stability and abundance by promoting its polyubiquitination, whereas OTUD4 acted to negate the effects of TRIM21-induced polyubiquitination, thereby stabilizing and increasing the levels of SHBs. Notably, TRIM21-mediated degradation of SHBs substantially impaired subviral particle and virion production and its biological activities such as migratory and angiogenic capabilities, opposite to the effect produced by the introduction of OTUD4. These findings suggest that TRIM21 and OTUD4 modulate SHBs protein stability and function through a ubiquitination-dependent proteasomal pathway, offering new insights into clearing SHBs and intervening in the progression of HBV-related liver diseases. IMPORTANCE: The small hepatitis B surface antigen (SHBs) is a key structural component of the hepatitis B virus (HBV) virion and subviral particles and is the most abundant HBV protein in individuals with chronic infection. Gaining a better understanding of its degradation pathways may help inform strategies to reduce SHBs levels and potentially support the design of targeted therapies. However, the specific mechanisms and processes involved in the degradation of SHBs remain largely unexplored. This study reveals that SHBs is degraded via the ubiquitin/proteasome pathway, specifically through K48-linked ubiquitination at the K122 site. TRIM21 promotes SHBs degradation by enhancing its polyubiquitination, while OTUD4 stabilizes SHBs by counteracting TRIM21’s effects. TRIM21 reduces SHBs stability, subviral particle and virion production, and its related biological activities, whereas OTUD4 stabilizes SHBs, promoting its accumulation. These findings highlight the roles of TRIM21 and OTUD4 in regulating SHBs stability and function, offering new insights into potential interventions for HBV-related liver diseases.

Persistent infection with hepatitis B virus (HBV) often leads to disruptions in lipid metabolism. Apolipoprotein AII (apoAII) plays a crucial role in lipid metabolism and is implicated in various metabolic disorders. However, whether HBV could regulate apoAII and contribute to HBV-related dyslipidemia and the underlying mechanism remain unclear. This study revealed significant reductions in apoAII expression in HBV-expressing cell lines, the serum, and liver tissues of HBV-transgenic mice. The impact of HBV on apoAII is related to small hepatitis B virus surface antigen (SHBs). Overexpression of SHBs decreased apoAII levels in SHBs-expressing hepatoma cells, transgenic mice, and the serum of HBV-infected patients, whereas suppression of SHBs increased apoAII expression. Mechanistic investigations demonstrated that SHBs repressed the apoAII promoter activity through a HNF4α- and C/EBPγ-dependent manner; SHBs simultaneously upregulated C/EBPγ and downregulated HNF4α by inhibiting the PI3K/AKT signaling pathway through activating endoplasmic reticulum (ER) stress. Serum lipid profile assessments revealed notable decreases in high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG) in SHBs-transgenic mice compared to control mice. However, concurrent overexpression of apoAII in these mice effectively counteracted these reductions in lipid levels. In HBV patients, SHBs levels were negatively correlated with serum levels of HDL-C, LDL-C, TC, and TG, whereas apoAII levels positively correlated with lipid content. This study underscores that SHBs contributes to dyslipidemia by suppressing the PI3K/AKT pathway via inducing ER stress, leading to altered expression of HNF4α and C/EBPγ, and subsequently reducing apoAII expression. IMPORTANCE: The significance of this study lies in its comprehensive examination of how the hepatitis B virus (HBV), specifically through its small hepatitis B virus surface antigen (SHBs), impacts lipid metabolism—a key aspect often disrupted by chronic HBV infection. By elucidating the role of SHBs in regulating apolipoprotein AII (apoAII), a critical player in lipid processes and associated metabolic disorders, this research provides insights into the molecular pathways contributing to HBV-related dyslipidemia. Discovering that SHBs downregulates apoAII through mechanisms involving the repression of the apoAII promoter via HNF4α and C/EBPγ, and the modulation of the PI3K/AKT signaling pathway via endoplasmic reticulum (ER) stress, adds critical knowledge to HBV pathogenesis. The research also shows an inverse correlation between SHBs expression and key lipid markers in HBV-infected individuals, suggesting that apoAII overexpression could counteract the lipid-altering effects of SHBs, offering new avenues for understanding and managing the metabolic implications of HBV infection.