Abstract Infectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are significant global threats to poultry health and production. While IBDV induces severe immunosuppression, undermining host defense and vaccine efficacy, H9N2 AIV is characterized by widespread prevalence, persistent shedding, and substantial economic losses. Conventional inactivated vaccines often fail to elicit robust cellular immunity and necessitate multiple booster doses, underscoring the urgent requirement for advanced multivalent vaccination platforms. To address this, we developed a recombinant herpesvirus of turkey (rHVT BAC-VP2-HA) using a bacterial artificial chromosome (BAC) vector system, engineered to co-express the major protective antigen VP2 of IBDV and the hemagglutinin ( HA ) of H9N2 AIV. Genetic stability and in vitro characterization confirmed that the recombinant exhibited replication kinetics and plaque morphology comparable to parental HVT, with stable antigen expression. In SPF chickens, rHVT BAC-VP2-HA induced strong humoral immune responses against both target antigens, comparable to those elicited by a commercial inactivated vaccine. Crucially, the recombinant virus significantly enhanced cellular immunity, evidenced by markedly elevated CD3 + CD8 + T cell responses. Upon challenge, the recombinant conferred high clinical protection (86%) against virulent IBDV, significantly ameliorating bursal pathology and reducing viral loads. Notably, it provided complete (100%) protection against H9N2 AIV, effectively abolishing viral shedding and suppressing viral replication in respiratory tissues. These results demonstrate that rHVT BAC-VP2-HA is a safe and efficacious candidate capable of eliciting humoral and cellular immune responses, offering a promising strategy for the integrated control of major poultry diseases. Importance Infectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are major pathogens that frequently co-circulate in poultry, where IBDV-induced immunosuppression compromises the efficacy of vaccination against other infectious diseases. Conventional inactivated vaccines primarily induce humoral immunity and are often insufficient to prevent viral shedding or provide broad protection against multiple pathogens. In this study, we developed a recombinant herpesvirus of turkeys (HVT) vaccine co-expressing the IBDV VP2 and H9N2 HA antigens and demonstrated that it induces both robust antibody responses and enhanced CD8 + T cell immunity. Notably, this vaccine not only provided effective protection against IBDV but also completely prevented viral shedding following H9N2 challenge. These findings highlight the advantage of HVT-vectored multivalent vaccines in eliciting balanced immune responses and controlling virus transmission, providing important insights for the development of next-generation vaccines against immunosuppressive and respiratory viral co-infections in poultry.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease in pigs, threatens swine production and can infect other mammals, with rare zoonotic cases reported. Effective antiviral agents against PRV remain lacking. Here, we constructed and purified a recombinant fusion protein combining porcine Interleukin-2 (IL-2), interferon α (IFN-α), and Thymosin α1 (Tα1). The fusion protein showed no cytotoxicity or induction of apoptosis in PK-15 cells. In vitro, it significantly enhanced peripheral blood mononuclear cell (PBMC) proliferation and upregulated expression of key interferon-stimulated genes (ISGs) involved in antiviral defense, including IFIT1, ISG15, MX1, and OAS, which are critical for antiviral activity. Analysis of the viral life cycle indicated that IL-2–IFN-α–Tα1 inhibited PRV replication without affecting viral attachment, entry, assembly, or egress. Treatment with the fusion protein conferred marked therapeutic efficacy against PRV infection, including improved survival, reduced viral loads in the brain and spleen, and alleviated inflammatory pathology. These results demonstrate that IL-2–IFN-α–Tα1 fusion protein has potential as a novel immunomodulatory agent for controlling PRV infection in swine, offering a promising strategy for antiviral intervention in veterinary medicine.

PD-L1, which is highly expressed on the surface of breast cancer cells, is one of the key mechanisms by which it achieves immune escape and promotes tumor progression by inhibiting the anti-tumor activity of T cells. In recent years, immunotherapy targeting PD-1/PD-L1 has received widespread attention in breast cancer treatment, so it is particularly important to deeply understand the mechanism of PD-L1 expression regulation. The regulation of PD-L1 expression mainly occurs at the levels of transcription, translation, and post-translational modification. In this paper, we reviewed the multiple post-translational modifications of PD-L1 known in breast cancer cells, including phosphorylation, ubiquitination, glycosylation, acetylation, etc., and explored the crosstalk between these modifications and their synergistic or antagonistic effects on PD-L1 function. By summarizing the complex regulatory network of PD-L1 post-translational modifications, this paper aims to provide new ideas and potential targets for the optimization of breast cancer immunotherapy.

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus whose productive infection and cell-to-cell spread rely on the formation of infectious mature virions, a process mediated by tegument proteins. UL16 is a conserved herpesviral tegument protein, in other herpesviruses, its deletion causes severe replication defects. However, its role in MDV remains unknown. In this study, a UL16-null MDV mutant was generated using BAC-based reverse genetics to investigate UL16’s function. Deletion of UL16 completely abrogated the recovery of infectious virus from BAC DNA in chicken embryonic fibroblasts, whereas genetic restoration or transient complementation of UL16 fully restored viral replication. The rescued viruses exhibited plaque morphology and growth kinetics indistinguishable from those of the parental virus. Transcriptional profiling showed that UL16 deletion did not significantly alter the expression of representative immediate-early, early, or late viral genes. In contrast, transmission electron microscopy revealed that although nuclear capsid assembly proceeded normally, cytoplasmic virion maturation was severely impaired in the absence of UL16, leading to a failure in secondary envelopment and the absence of fully enveloped virions. In summary, these results demonstrate that UL16 is indispensable for MDV replication in vitro and plays a critical role in the late stages of virion maturation, offering new insights into the molecular mechanisms governing MDV assembly.

Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces severe immunosuppression and T-cell lymphomas in chickens, posing a major threat to poultry production. Viral morphogenesis in alphaherpesviruses depends on coordinated membrane remodeling processes mediated by conserved viral membrane proteins. Although MDV encodes a homolog of UL20 protein, its functional role remains unknown. In this study, we employed an integrated approach combining bioinformatic analysis, bacterial artificial chromosome (BAC)-based mutagenesis, and ultrastructural characterization to elucidate the role of MDV UL20 in viral replication and morphogenesis. Sequence analysis revealed that MDV UL20 is a conserved four-pass transmembrane protein with a membrane topology similar to that of UL20 homologs in other alphaherpesviruses. To assess its functional relevance, a UL20 deletion mutant (Md5BACΔUL20) was generated, and its biological properties were evaluated in chicken embryonic fibroblasts. Deletion of UL20 completely abolished the production of infectious virus, resulting in the absence of plaque formation and impaired cell-to-cell spread. In contrast, genetic reconstitution of UL20 fully restored viral replication to wild-type levels. Transmission electron microscopy demonstrated that UL20 deletion did not affect nuclear capsid assembly but markedly reduced the number of cytoplasmic capsids, secondary envelopment intermediates, and extracellular virion accumulation. UL20 re-expression assays confirmed that these defects were specifically attributable to the loss of UL20. Collectively, these findings demonstrate that MDV UL20 is dispensable for nuclear capsid formation but essential for cytoplasmic secondary envelopment and infectious virion production, providing new insights into the membrane-associated mechanisms underlying MDV morphogenesis.

Marek's disease virus (MDV), a highly oncogenic alphaherpesvirus of chickens, causes severe immunosuppression and fatal T-cell lymphomas. The UL24 gene is conserved among alphaherpesviruses and contributes to viral replication and pathogenesis in several members of this subfamily; however, its role in MDV remains undefined. Here, we constructed a UL24-deletion mutant (Md5BACΔUL24) and its repaired revertant (Md5BACΔUL24-Re) in the virulent Md5 strain using Red-mediated recombination. In vitro, UL24 deletion significantly impaired viral replication, as evidenced by smaller plaque size and reduced viral genome copies compared to parental and revertant viruses. In specific pathogen-free (SPF) chickens, UL24 deficiency markedly decreased viral loads in the spleen, prevented lymphoid organ atrophy, and abolished tumor development, indicating a profound attenuation of virulence. Bioinformatic analyses revealed a putative nuclear localization signal and a conserved PD-(D/E)XK endonuclease motif within MDV UL24, suggesting involvement in nuclear processes critical for replication. Collectively, these findings establish UL24 as a critical role of MDV replication and pathogenesis and represent a promising target for the rational design of attenuated or recombinant MDV vaccine vectors.

Background N6-methyladenosine, the most prevalent post-transcriptional modification in eukaryotes, plays a critical role in regulating gene expression and disease pathogenesis. Concurrently, G-quadruplex structures are implicated in key biological processes, including the regulation of transcription and post-transcriptional events. Emerging evidence suggests that m6A modifications and G4 structures colocalize within viral genomes and human pre-mRNAs; however, their functional interplay remains poorly understood. Results Using existing MeRIP-seq data from Marek's disease virus (MDV )-infected samples, we investigated the colocalization pattern and its potential role in viral infection. Our bioinformatic analyses revealed that the predicted G4 structures were predominantly two G-tetrad G-quadruplexes. Genes with m6A-G4 colocalization were significantly enriched in pathways related to immune response and tumorigenesis. Then we verified the presence of co-localization on the CCL4, which inhibited CCL4 expression. Co-localization also affected viral replication and regulated ICP4 expression. Conclusions We systematically characterized the features of G4 structures co-localized with m6A modifications during MDV infection, investigated the role in viral replication and gene regulation, and provided experimental evidence for m6A-G4 co-localization. These findings shed new light on the epitranscriptomic regulatory mechanisms employed by an oncogenic virus and may inform the development of novel antiviral and anti-tumor therapeutic strategies

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus causing rapid onset of malignant T-cell lymphomas in chickens. UL23-encoded thymidine kinase (TK) has highly conserved sequences in distinctive alphaherpesviruses. However, its enzymatic activity in viral replication and pathogenesis is poorly understood. Here, we found that the nucleotide-binding sites and the functional domains related to activity of TK from different alphaherpesviruses are strongly conserved. We show that an MDV-1 UL23-null mutation (Md5BACΔUL23) significantly reduces MDV replication in vitro and in vivo. Interestingly, chimeric viruses with replacement of MDV-1 UL23 with MDV-2, HVT, PRV, or HSV-1 UL23 showed partial recovery of MDV replication and pathogenicity. In addition, Md5BACΔUL23 infection resulted in higher survival rate and lower MDV-specific tumor incidence, which could be partially compensated by chimeric viruses. The replication properties of UL23 chimeric alphaherpesviruses are susceptible to acyclovir inhibition, whereas Md5BACΔUL23 exhibits complete resistance. Overall, our establishment of the MDV-TK chimeric model provides a robust basis for evaluating TK-targeted therapeutics, accelerating clinical translation of novel anti-herpesvirus strategies.

Methyltransferase-like-3 (METTL3)-mediated N6-methyladenosine (m6A) modification of messenger RNAs plays a pivotal role in regulating innate immune responses, either promoting or combating virus replication. However, the biological function of METTL3 during porcine reproductive and respiratory syndrome virus (PRRSV) infection remains unclear. In this study, we found that PRRSV infection reprograms m6A modifications in cellular transcripts, enhances METTL3 expression, and alters its subcellular distribution. Intriguingly, METTL3 overexpression facilitates PRRSV replication, whereas its deficiency suppresses it, primarily through the negative regulation of type I interferon (IFN-I) production. Further investigation revealed that METTL3 interacts with and promotes the degradation of IκB kinase-ε (IKKε) during PRRSV infection. Mechanistically, METTL3-mediated m6A modification of SQSTM1 (sequestosome 1) enhances SQSTM1 messenger RNA (mRNA) expression, increasing autophagy levels. Moreover, METTL3 facilitates the formation of K63-linked ubiquitin chains on IKKε, targeting it for degradation via SQSTM1-dependent selective autophagy. Collectively, our findings unveil a novel mechanism whereby METTL3 facilitates PRRSV replication by suppressing antiviral innate immunity, thereby offering potential targets for antiviral therapy.IMPORTANCEPorcine reproductive and respiratory syndrome (PRRS), induced by the porcine reproductive and respiratory syndrome virus (PRRSV), poses a highly contagious threat to the global swine industry, leading to substantial economic losses. The genetic variability and immune evasion capabilities of PRRSV complicate the development of effective vaccines and control strategies. Thus, a comprehensive understanding of PRRSV's immune evasion mechanisms is imperative. In this study, we reveal that METTL3 plays a pivotal role in PRRSV's evasion of interferon (IFN) immunity. Specifically, METTL3 targets IKKε, inducing its autophagy degradation and subsequently inhibiting the expression of interferon beta 1 (IFNB1). Furthermore, PRRSV infection alters the N6-methyladenosine (m6A) modification of various host genes, with notable changes observed in the m6A modification and transcriptional levels of SQSTM1, which are regulated by METTL3. This regulation is crucial for SQSTM1-mediated autophagy degradation of IKKε. Our findings offer novel insights into the mechanisms underlying host protein involvement in PRRSV's immune evasion.

African Swine Fever (ASF) is a highly infectious, acute, hemorrhagic Swine disease caused by the African Swine Fever Virus (ASFV). It is lethal to both domestic pigs and wild boars, and has caused significant economic losses to the global pig industry. Vaccines represent the most significant means of preventing and treating viral diseases. However, due to the complex genome and strong immune evasion mechanism of ASFV, there has been no successful development of a vaccine that has been made commercially available on a global scale. Thus, the focus of present research efforts is the study of its viral protein. B117L is a non-structural protein encoded by the ASFV, the function of which has not yet been fully defined. It belongs to a variable region in the viral genome. Direct studies on the B117L protein are relatively scarce, but analogous proteins (MGF360/505 family) have been found to have immunoregulatory functions. B117L may be a potential immunoregulatory protein of ASFV involved in viral immune evasion, replication regulation, or pathogenesis, but further investigation is required to determine its specific function. The amino acid sequence of African swine fever B117L protein is as follows: MGYTIQLDGDYCWDEDPTHHDPYMQANATSHVATSYATTSHAATPHAAAHHTFHEPFIKLNLTDKNIFNGLGFILIVIFIYLLLITLQQMLTRHIYNTVQHCVKAHLDSKNLQ. In order to facilitate a more effective expression of the B117L protein, the hydrophobic side B117L - 213 in amino acid protein is utilized, and its amino acid sequence is MGYTIQLDKDGDYCWDEDPTHHDPYMQANATSHVATSYATTSHAATPHAAAHHTFHEPFIKLNLTDKNIFN. The preparation of B117L-213 monoclonal antibody has the potential to facilitate further understanding of the B117L protein, the development of an ASFV vaccine, and the control of ASFV epidemics. The study encompassed of three strains of ASFV B117L-213 protein-specific monoclonal antibodies: 7F8D2A6, 8B11C8F9 and 8H1D5H8 (IgG2b/Kappa subtypes). The findings of this study are of considerable significance for the design and development of ASFV vaccines, and also provide relevant information for the study of the function of the B117L protein.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, febrile, highly contagious, and lethal disease that poses a severe threat to the global pig farming industry. Currently, no globally recognized, safe, and effective commercial ASF vaccine has been developed, making vaccination a crucial strategy for outbreak control. The ASFV structural proteins p72, p30, and p54 are key targets for vaccine development. In this study, we developed a novel baculovirus vector-based system for surface display of a recombinant protein comprising epitopes from p72, p30, and p54. Upon infection, the recombinant protein was expressed and anchored on the plasma membrane of Sf-9 cells. Purified virus analysis revealed that the Bac-recombinant protein enhanced gene delivery and transgene expression in mammalian cells compared to the Bac-Wild Type (Bac-WT). In a murine model, the Bac-recombinant protein induced significantly higher IFN-γ and IL-4 levels than Bac-p30 and the negative control. However, further evaluation in swine models is required to confirm its protective potential against ASFV. Furthermore, it also elicited a robust antibody response, generating high-titer Bac-recombinant protein-specific antibodies. Therefore, these findings suggest that the ASFV Bac-recombinant protein is a promising candidate for a vector-based vaccine.

Background:Myeloid Zinc Finger 1 (MZF1) is a zinc finger transcription factor gene that regulates gene expression by recognizing and binding to specific DNA sequences. Preliminary studies have suggested that MZF1 plays a pivotal role in the invasion and metastasis of various solid cancers. However, its role within the tumor immune microenvironment, as well as its prognostic value and potential for predicting responses to immunotherapy across different cancer types, remains inadequately explored and warrants a comprehensive systematic analysis. Methods:MZF1 expression levels in various cancers were obtained from the Cancer Genome Atlas (TCGA) database. The TISCH web tool analyzed MZF1 expression in 32 cell types. A spatial distribution map of MZF1 related to cancer tissue markers was created using the STOmics DB. A univariate Cox regression analysis was performed to evaluate MZF1's prognostic value. The cBioPortal database helped explore potential MZF1 mutations across cancer types. The TIMER2.0 database was used to study the relationship between MZF1 expression and immune cell infiltration. Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed to elucidate signaling pathways modulated by MZF1. Drug sensitivity testing for MZF1 was done using the CellMiner, the Cancer Therapeutics Response Portal (CTRP), and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Finally, MZF1 knockdown was achieved with siRNA silencing. Results:Changes in MZF1 expression are linked to the prognosis of most cancer patients. In the tumor microenvironment, MZF1 is mainly found in CD4 Tconv cells and monocytes/macrophages. Studies show that MZF1 is associated with cancer immunotherapy markers, immune cell infiltration, and immune modulators. Additionally, its role in immune regulation was confirmed through analysis of StromalScore, ImmuneScore, ESTIMATE, and immune infiltration. Molecular docking identified MZF1-targeted drugs, with validated effects on breast cancer and gastric cancer cell survival and migration in vitro. Lastly, the knockdown of MZF1 can suppress cancer cell migration. Conclusion:Collectively, these findings underscore the pivotal role of MZF1 in tumor biology and immune modulation. MZF1 emerges as a promising prognostic biomarker and potential therapeutic target, offering novel avenues for cancer treatment strategies.

During human herpesvirus infection, dynamic alterations of N6-methyladenosine (m6A) modification have been extensively observed in viral and cellular transcriptomes. This modification plays a crucial role in RNA metabolism, serving as a novel regulator of gene expression alongside DNA and protein modifications. Notably, reversible changes in a single m6A modification site can impact viral replication and pathogenicity. Recent studies have reported changes in m6A modification-associated epitranscriptomes and their functional analysis during animal herpesvirus infections. This review focuses on the research progress of m6A modification on the transcriptome in both human and animal herpesvirus infections within the same family. Specifically, it examines the dynamic alterations of m6A modification-associated epitranscriptomes, the expression of m6A-machinery proteins, regulatory molecular mechanisms associated with herpesvirus infection, and potential clinical applications. By addressing the gaps in research on m6A modification in animal viruses, new insights into the regulatory molecular mechanisms of viral diseases may be uncovered. Furthermore, natural hosts infected with animal herpesvirus serve as valuable biomedical models for studying the regulation of m6A modification on viral replication and pathogenesis, thereby supporting the development of novel vaccine and drug targets.

RIN3, a member of the RIN protein family, plays a pivotal role in disease progression by modulating Rab5 activity and influencing cell signaling pathways, which in turn affect tumor proliferation and migration. Our study systematically examined RIN3 expression in various tumor types using data from TCGA, GTEx, and single-cell RNA sequencing of 77 tumor types in the TISCH database, aiming to clarify its potential role in cancer. We evaluated the association between RIN3 levels and patient survival via univariate Cox regression and analyzed its correlation with immune cell infiltration using TIMER2.0. Additionally, GSEA and GSVA were employed to explore the involvement of RIN3 in immune responses and metabolic processes, while molecular docking and bioinformatics approaches predicted its interactions with anticancer drugs. Functional assays in breast cancer models confirmed that downregulation of RIN3 significantly inhibited cell proliferation and migration. Our results revealed considerable variations in RIN3 expression across tumors, an inverse relationship with CNVs and DNA methylation, and a significant correlation with immunotherapy biomarkers. These findings suggest RIN3 as a promising biomarker and therapeutic target, particularly for BRCA-mutated cancers, and may guide the development of novel anticancer strategies.

BackgroundThe RAB5B protein belongs to the RAB family and is primarily localized to early endosomes. It regulates the endocytic pathway through its GTPase activity, thereby affecting various aspects such as cell signaling and metabolic regulation. Dysfunction of RAB5B is closely associated with the progression and deterioration of multiple types of tumors. Although studies have revealed the functional mechanisms of RAB5B in specific tumor types, its role in pan-cancer and the underlying molecular mechanisms still lack in-depth analysis.MethodsA comparative analysis of RAB5B gene expression was conducted using transcriptomic datasets from Cancer Genome Map (TCGA) and Genotype-Tissue Expression (GTEx), followed by tissue distribution profiling via Human Protein Atlas (HPA) and GeneMANIA to map its expression across human tissues. The TISCH database identified primary cell types expressing RAB5B within the tumor microenvironment, while univariate Cox regression modeling evaluated its prognostic significance in cancer outcomes. Integrative genomic analyses using cBioPortal and Gene Set Cancer Analysis (GSCA) further characterized RAB5B’s genomic alterations and cancer-specific profiles. Gene_DE module 2.0 (TIMER 2.0) deciphered associations between RAB5B expression and tumor-infiltrating immune cell subsets. To elucidate functional mechanisms, Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) identified biological pathways co-regulated with RAB5B, and in silico approaches combining CellMiner with molecular docking predicted interactions between RAB5B and anticancer drugs. In vitro wound healing assays were performed to validate RAB5B’s role in modulating cellular migration dynamics, complementing bioinformatics findings with experimental evidence.ResultsRAB5B protein expression varied significantly across tumors, with different prognostic values. In most malignancies, RAB5B expression correlated positively with Copy Number Variation (CNV) and methylation. It also correlated significantly with immunotherapy biomarkers and responses. ESTIMATE and immune infiltration analyses highlighted RAB5B’s link to immunosuppression, emphasizing its role in immune regulation. Molecular docking and experimental validation showed that downregulating RAB5B inhibited cell proliferation and reduced cancer cell migration.ConclusionOur study revealed the key role of RAB5B in tumor biomarkers. RAB5B inhibits ectopic metastasis of tumor cells mainly by regulating the process of cell adhesion and migration. This discovery is of great significance for developing new anticancer inhibitors.

AIMS:Previous studies indicate that MAF BZIP Transcription Factor F (MAFF) facilitates ectopic metastasis and tumor cell migration. While its role in neoplasm progression is recognized, a thorough pan-cancer analysis of MAFF's impact remains pending. MAIN METHODS:MAFF expression across normal and tumor tissues was analyzed using transcriptomic data from Genomic Data Commons (GDC) and UCSC XENA, with protein details from Human Protein Atlas (HPA) and GeneMANIA. Tumor Immune Single-cell Hub (TISCH) and Spatial Transcriptomics Omics DataBase (STOmics DB) identified MAFF expression in the tumor microenvironment (TME). MAFF's prognostic significance and immune-related gene associations were evaluated through univariate Cox regression, TIMER2.0 immune cell infiltration analysis, and Spearman correlation. Critical pathways were identified using Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA), while molecular docking explored anticancer agent interactions. KEY FINDINGS:MAFF expression varies across cancers, affecting tumor prognosis, notably in monocytes/macrophages and endothelial cells. Copy number variation (CNV) positively correlates with MAFF expression, while methylation shows inverse correlation. MAFF mutations significantly affect LGG patient prognosis and correlate with immune therapy responses. ESTIMATE and immune profiling linked MAFF to immunosuppression pathways. Molecular docking identified MAFF-targeted drugs, with validated effects on breast cancer and endometrial cancer cell survival and migration in vitro. SIGNIFICANCE:Multi-omics analysis identified MAFF as a potential prognostic marker correlating with tumor immunity and microenvironment, suggesting its value for personalized cancer immunotherapy, particularly in BRCA and UCEC.

GMIP, a member of the RhoGAP family, plays a critical role in cytoskeletal remodelling, cell migration and immune modulation. Its aberrant expression in cancers suggests a pivotal role in tumour progression. GMIP expression was assessed using transcriptomic datasets from GDC and UCSC XENA, and protein distribution across tissues via HPA and GeneMANIA. The TISCH database identified primary GMIP-expressing cell types in the tumour microenvironment. Univariate Cox regression assessed GMIP's prognostic potential, while cBioPortal and GSCA explored genomic alterations. TIMER 2.0 was used to investigate immune cell infiltration and GMIP's role in immune regulation. GSEA and GSVA unveiled GMIP-related biological pathways, and molecular docking with CellMiner identified potential drug interactions. In vitro assays confirmed GMIP's functional relevance in breast cancer. GMIP exhibits differential expression across multiple cancer types, demonstrating significant prognostic implications. Its expression is inversely correlated with CNV and methylation in several cancers. GMIP is closely linked to immunotherapy biomarkers and immune suppression, influencing therapeutic responses. Functional studies suggest that GMIP inhibition reduces cancer cell proliferation and migration. GMIP is identified as a promising oncological biomarker, particularly in breast cancer, with potential therapeutic implications. GMIP's therapeutic potential is especially pronounced in BRCA-mutated tumours, underscoring its relevance for novel anticancer interventions.

Marek’s disease virus (MDV)-encoded US3 is a highly conserved serine/threonine protein kinase in alpha-herpesviruses. In other alpha-herpesviruses, such as pseudorabies virus (PRV), US3 phosphorylates the N6-methyladenosine (m6A) methyltransferase Wilms tumor 1-associated protein (WTAP), inhibiting m6A modification. However, the role and mechanism of US3-mediated WTAP phosphorylation during MDV infection remain undefined. Our study revealed that MDV infection in vitro does not alter WTAP expression, while significant changes in WTAP expression occur during the MDV life cycle in vivo. We demonstrated that MDV-encoded US3 interacts with and co-localizes with WTAP in the nucleus. Further analysis showed that US3 binds to WTAP's C-terminal domain and phosphorylates WTAP at S273, S305, S314, and S375. Notably, the interaction between US3 and WTAP does not affect WTAP stability but inhibits transcriptomic m6A modification and cellular protein translation. Therefore, these findings enhance our understanding of the molecular mechanisms underlying MDV infection.

Eukaryotic translation initiation factor 4A3 (eIF4A3)-mediated RNA metabolism is essential for cellular homeostasis and viral replication. However, its role in regulating antiviral innate immunity during pseudorabies virus (PRV) infection remains unknown. Here, we demonstrate that eIF4A3 protein expression was significantly downregulated both in vitro and in vivo during PRV infection. Functional assays showed that eIF4A3 overexpression suppressed PRV replication, whereas its knockdown enhanced viral replication. Mechanistically, eIF4A3 inhibited m6A modification of STING mRNA, thereby increasing its stability. Elevated STING protein expression promoted type I interferon (IFN-β) production via activation of the cGAS-STING signaling pathway. In summary, this study identifies a new antiviral mechanism in which eIF4A3 enhances innate immunity against PRV infection by regulating the stability of STING mRNA through modulating its m⁶A modification.

The ERK-mediated phosphorylation of the core m6A methyltransferase METTL3 has been linked to the regulation of embryonic stem cell differentiation and tumorigenesis. However, its role in the antiviral innate immune response remains unclear. In this study, we found that during infection with the prototypical alpha-herpesvirus Pseudorabies virus (PRV), ERK2 protein expression increased significantly, while METTL3 expression decreased both in vitro and in vivo. Overexpressing ERK2 and METTL3 effectively reduced PRV replication, while their knockdown promoted viral replication. The C-terminal domain and enzymatic active site of METTL3 were essential for suppressing viral replication. Mechanistically, ERK2 phosphorylates METTL3 at serine 43. We further found that ERK2-mediated phosphorylation at this site enhances the type I interferon (IFN-β)-induced innate immune response by activating the NF-κB pathway, increasing m6A modification, and elevating protein translation levels. Notably, combined treatment with ERK2 and METTL3 inhibitors promoted viral replication, intensified organ damage, and hastened mortality in mice by suppressing IFN-β production. In conclusion, our study reveals phosphorylation-dependent crosstalk between MAPK signaling and the m6A machinery in antiviral defense, identifies Ser43 as a functional hotspot for METTL3's immunoregulatory activity, and indicates that the ERK-METTL3 axis is a novel regulator of the antiviral innate immune response during alpha-herpesvirus infection. This work establishes a paradigm shift in understanding how post-translational modifications of RNA-modifying enzymes orchestrate antiviral immunity, providing new avenues for host-directed antiviral strategies.