[This corrects the article DOI: 10.1371/journal.ppat.1009294.].

The global outbreak of mpox caused by the mpox virus (MPXV) in 2022 and 2024 underscores the urgent need to elucidate mechanisms governing viral replication during pathogenesis. Metabolic reprogramming is a conserved hallmark of viral infections, however, the precise mechanisms by which MPXV manipulates host cell metabolism remain unknown. Here, it is demonstrated that MPXV hijacks aerobic glycolysis via lysine crotonylation of its I3 protein, which is essential for MPXV replication. Mechanistically, MYST histone acetyltransferase 1 (MYST1), an acetyltransferase upregulated by MPXV, binds to and catalyzes the crotonylation of I3. The crotonylated I3 interacts with WD-repeat protein 26 (WDR26) to prevent its ubiquitination-dependent degradation, leading to enhanced aerobic glycolysis and promoting MPXV replication. Either pharmacological inhibition of MYST1 using MC4033 or blocking aerobic glycolysis with the glycolytic inhibitors 2-Deoxy-D-glucose (2-DG) or dichloroacetic acid (DCA) effectively suppresses MPXV replication. These findings uncover a novel crotonylation-dependent mechanism through which MPXV reprograms host metabolism to facilitate viral propagation, and identify lysine crotonylation and aerobic glycolysis as potential therapeutic targets against mpox.

Although a fraction of functional peptides concealed within long non-coding RNAs (lncRNAs) is identified, it remains unclear whether lncRNA-encoded peptides are involved in the malignancy of cervical cancer (CC). Here, a 92-amino acid peptide is discovered, which is named TUBORF, encoded by lncRNA TUBA3FP and highly expressed in CC tissues. TUBORF inhibits ferroptosis to promote the malignant proliferation of CC cells. Mechanistically, human papillomavirus (HPV) oncogenes E6 and E7 upregulate TUBORF through CREB-binding protein (CBP)/E1A-binding protein p300 (p300)-mediated histone H3 lysine 27 acetylation (H3K27ac) of lncTUBA3FP enhancer. Furthermore, E6 and E7 elevate and recruit acetyltransferase establishment of sister chromatid cohesion N-acetyltransferase 1 (ESCO1) to bind to and acetylate TUBORF, which facilitates the degradation of immunity-related GTPase Q (IRGQ) via a ubiquitin-proteasome pathway, resulting in the inhibition of ferroptosis and promotion of the malignant proliferation of CC cells. Importantly, silencing ESCO1 or TURORF amplifies anticancer effects by paclitaxel both in CC cells and in vivo. These novel findings reveal oncopeptide TUBORF and its acetyltransferase ESCO1 as important regulators of ferroptosis and tumorigenesis during cervical cancer pathogenesis and establish the scientific basis for targeting these molecules for treating CC.

Spinal cord injury (SCI) is a severe neurological disorder that significantly impacts patients’ quality of life. Following SCI, the blood-spinal cord barrier (BSCB) is destroyed, leading to ischemia and hypoxia, which further exacerbates the imbalance in the spinal cord microenvironment. A2-type astrocytes, which arise under ischemic and hypoxic conditions, have been reported to promote SCI repair. However, the roles of exosomes derived from A2 astrocytes (A2-Exos) in SCI have not been explored. This study aims to investigate the role of A2-Exos in SCI repair, particularly in BSCB restoration, and to elucidate its potential mechanisms. GEO database analysis, western blotting, and immunofluorescence were used to detect A2 astrocyte polarization after SCI in mice. In vitro, A2 astrocytes were obtained through hypoxia induction, and A2-Exos were extracted via ultracentrifugation. An in vivo SCI model and a series of in vitro experiments demonstrated the reparative effects of A2-Exos on BSCB following SCI. Furthermore, miRNA sequencing analysis and rescue experiments confirmed the role of miRNAs in A2-Exos-mediated BSCB repair. Finally, luciferase assays and western blotting were performed to investigate the underlying mechanisms. The results showed that A2-Exos promote motor function recovery and BSCB repair in mice following SCI. In vitro, A2-Exos facilitated BSCB reconstruction and endothelial cell autophagy. miRNA sequencing identified miR-5121 as the most significantly enriched miRNA in A2-Exos, suggesting its involvement in BSCB repair and autophagy regulation. AKT2 was identified as a potential downstream target of miR-5121. Functional gain- and loss-of-function experiments further validated the miR-5121/AKT2 axis. Finally, we demonstrated that the AKT2/mTOR/p70S6K pathway may mediate the effects of miR-5121 in A2-Exos on BSCB repair.

RNA N6-methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) plays a crucial role in regulating innate immunity. Lysine acylation, a widespread protein modification, influences protein function, but its impact on ALKBH5 during viral infections has not been well characterized. This study investigates the presence and regulatory mechanisms of a previously unidentified lysine acylation in ALKBH5 and its role in mediating m6A modifications to activate antiviral innate immune responses. We demonstrate that ALKBH5 undergoes lactylation, which is essential for an effective innate immune response against DNA herpesviruses, including herpes simplex virus type 1 (HSV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), and mpox virus (MPXV). This lactylation attenuates viral replication. Mechanistically, viral infections enhance ALKBH5 lactylation by increasing its interaction with acetyltransferase ESCO2 and decreasing its interaction with deacetyltransferase SIRT6. Lactylated ALKBH5 binds interferon-beta (IFN-β) messenger RNA (mRNA), leading to demethylation of its m6A modifications and promoting IFN-β mRNA biogenesis. Overexpression of ESCO2 or depletion of SIRT6 further enhances ALKBH5 lactylation to strengthen IFN-β mRNA biogenesis. Our results identify a posttranslational modification of ALKBH5 and its role in regulating antiviral innate immune responses through m6A modification. The finding provides an understanding of innate immunity and offers a potential therapeutic target for HSV-1, KSHV, and MPXV infections.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent genetic disorder affecting the kidneys. Understanding epigenetic regulatory mechanisms and the role of microRNAs (miRNAs) is crucial for developing therapeutic interventions. Two mRNA datasets (GSE7869 and GSE35831) and miRNA expression data (GSE133530) from ADPKD patients were used to find differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), with a focus on genes regulated by hub transcription factors (TFs) and their target genes. The expression of hub TFs was validated in human kidneys and animal models through Western Blot (WB) and RT-PCR analysis. The location of the hub TF proteins in kidney cells was observed by a laser confocal microscope. A total of 2037 DEGs were identified. DEM analysis resulted in 59 up-regulated and 107 down-regulated miRNAs. Predicted target DEGs of DEMs indicated two top dysregulated TFs: hepatocyte nuclear factor 4 alpha (HNF4α) and Kruppel-like factor 4 (KLF4). RT-PCR, WB, and immunochemistry results showed that mRNA and protein levels of HNF4α were significantly decreased while KLF4 levels were significantly up-regulated in human ADPKD kidneys and Pkd1 conditional knockout mice compared with normal controls. Laser confocal microscopy revealed that KLF4 was mainly located in the cytoplasm while HNF4α was in the nucleus. Functional enrichment analysis indicated that genes regulated by HNF4α were mainly associated with metabolic pathways, while KLF4-regulated genes were linked to kidney development. Drug response prediction analysis revealed potential drug candidates for ADPKD treatment, including BI-2536, Sepantronium, and AZD5582. This integrated analysis provides new epigenetic insights into the complex miRNA-TF-mRNA network in ADPKD and identifies HNF4α and KLF4 as key TFs. These findings offer valuable resources for further research and potential drug development for ADPKD.

N4-acetylcytidine (ac4C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac4C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac4C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac4C level of tRNASer-CGA-1-1. Mutagenesis at the ac4C site in tRNASer-CGA-1-1 inhibited its ac4C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNASer-CGA-1-1 ac4C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac4C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases.

Ferroptosis, a defensive strategy commonly employed by the host cells to restrict pathogenic infections, has been implicated in the development and therapeutic responses of various types of cancer. However, the role of ferroptosis in oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV)-induced cancers remains elusive. While a growing number of non-histone proteins have been identified as acetylation targets, the functions of these modifications have yet to be revealed. Here, we show KSHV reprogramming of host acetylation proteomics following cellular transformation of rat primary mesenchymal precursor. Among them, SERPINE1 mRNA binding protein 1 (SERBP1) deacetylation is increased and required for KSHV-induced cellular transformation. Mechanistically, KSHV-encoded viral interleukin-6 (vIL-6) promotes SIRT3 deacetylation of SERBP1, preventing its binding to and protection of lipoyltransferase 2 (Lipt2) mRNA from mRNA degradation resulting in ferroptosis. Consequently, a SIRT3-specific inhibitor, 3-TYP, suppresses KSHV-induced cellular transformation by inducing ferroptosis. Our findings unveil novel roles of vIL-6 and SERBP1 deacetylation in regulating ferroptosis and KSHV-induced cellular transformation, and establish the vIL-6-SIRT3-SERBP1-ferroptosis pathways as a potential new therapeutic target for KSHV-associated cancers.

Post-translational modifications (PTMs) are essential for host antiviral immune response and viral immune evasion. Among a set of novel acylations, lysine propionylation (Kpr) has been detected in both histone and non-histone proteins. However, whether protein propionylation occurs in any viral proteins and whether such modifications regulate viral immune evasion remain elusive. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) can be propionylated in lysine residues, which is required for effective inhibition of IFN-β production and antiviral signaling. Mechanistically, vIRF1 promotes its own propionylation by blocking SIRT6's interaction with ubiquitin-specific peptidase 10 (USP10) leading to its degradation via a ubiquitin-proteasome pathway. Furthermore, vIRF1 propionylation is required for its function to block IRF3-CBP/p300 recruitment and repress the STING DNA sensing pathway. A SIRT6-specific activator, UBCS039, rescues propionylated vIRF1-mediated repression of IFN-β signaling. These results reveal a novel mechanism of viral evasion of innate immunity through propionylation of a viral protein. The findings suggest that enzymes involved in viral propionylation could be potential targets for preventing viral infections.

N -acetyltransferase 10 (NAT10) is an N 4 ‐acetylcytidine (ac 4 C) writer that catalyzes RNA acetylation at cytidine N 4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac 4 C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac 4 C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac 4 C sites in PAN RNA in the context of KSHV infection abolishes PAN ac 4 C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac 4 C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac 4 C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac 4 C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac 4 C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.

While the application of early screening and HPV vaccines has reduced the incidence and mortality rates of cervical cancer, it remains the third most common carcinoma and fourth leading cause of cancer-associated death among women worldwide. The precise mechanisms underlying progression of cervical cancer are not fully understood at present. Here, we detected significant down-regulation of 15-hydroxyprostaglandin dehydrogenase (HPGD) in cervical cancer tissues. Overexpression of HPGD inhibited cervical cancer cell proliferation, migration and anchorage-independent growth to a significant extent. To clarify the mechanisms underlying HPGD down-regulation in cervical cancer, miRNA microarray, bioinformatics and luciferase reporter analyses were performed. HPGD was identified as a direct target of miR-146b-3p displaying up-regulation in cervical cancer tissues. Similar to the effects of HPGD overexpression, down-regulation of miR-146b-3p strongly suppressed proliferation, migration and anchorage-independent growth of cervical cancer cells. Furthermore, HPGD negatively regulated activities of STAT3 and AKT that promote cervical cancer cell proliferation. Notably, HPV oncogenes E6 and E7 were determined as potential contributory factors to these alterations. Our results collectively suggest that the HPGD/miR-146b-3p axis plays a significant role in cervical cancer and may serve as a potentially effective therapeutic target.

Abstract Immunosuppressive neutrophils are identified as a specialized heterogeneous population in cancer patients that accelerate tumor progression. However, the developmental stage and the source of this population remain elusive. To address this issue, we unified three reported neutrophil nomenclatures and demonstrated that immature neutrophils predominate tumor tissues of patients. On the quest to identify the origin of immature neutrophils, we unexpectedly discovered that immature neutrophils collected from the bone marrow of healthy people and mice reconstituted with human immune cells (HIS mice) are naturally immunosuppressive and tumor-promoting, whereas the mouse counterpart is not. Single-cell comparison of immature neutrophils isolated from human tumors, human bone marrow and HIS mouse bone marrow samples reveal FCN1 as a common marker for this heterogeneous population. To further investigate the link between peripheral and bone marrow immature neutrophils, we created a novel immunodeficient strain, to overcome the poor human neutrophil engraftment in the periphery. HIS mice based on this strain manifest similar kinetics of immature neutrophil expansion as cancer patients both in the bone marrow and periphery after tumor cell inoculation. Finally, we proved that eliminating bone marrow immature neutrophils delays tumor growth in HIS mice. In summary, human immunosuppressive neutrophils are predominantly immature neutrophils and targeting this population in bone marrow may represent a novel strategy for cancer immunotherapy. Citation Format: Wei Liu, Keying Che, Chun Lu, Jia Wei, Yan Li. Identification of immunosuppressive and tumor-promoting neutrophils in human bone marrow and a novel mouse model that supports human neutrophil engraftment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2878.

Reprogramming of host metabolism is a common strategy of viral evasion of host cells, and is essential for successful viral infection and induction of cancer in the context cancer viruses. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer caused by KS-associated herpesvirus (KSHV) infection. KSHV-encoded viral interferon regulatory factor 1 (vIRF1) regulates multiple signaling pathways and plays an important role in KSHV infection and oncogenesis. However, the role of vIRF1 in KSHV-induced metabolic reprogramming remains elusive. Here we show that vIRF1 increases glucose uptake, ATP production and lactate secretion by downregulating heterogeneous nuclear ribonuclear protein Q1 (hnRNP Q1). Mechanistically, vIRF1 upregulates and recruits E3 ubiquitin ligase Kelch-like 3 (KLHL3) to degrade hnRNP Q1 through a ubiquitin-proteasome pathway. Furthermore, hnRNP Q1 binds to and stabilizes the mRNA of glycerophosphodiester phosphodiesterase domain containing 1 (GDPD1). However, vIRF1 targets hnRNP Q1 for degradation, which destabilizes GDPD1 mRNA, resulting in induction of aerobic glycolysis. These results reveal a novel role of vIRF1 in KSHV metabolic reprogramming, and identifying a potential therapeutic target for KSHV infection and KSHV-induced cancers.

Kaposi's sarcoma‐associated herpesvirus (KSHV) causes life‐long latent infection and malignancies, including KS commonly found in AIDS patients. Lytic replication can be induced to kill tumor cells harboring latent KSHV, through viral cytopathic effects and the subsequent antiviral immune responses. Viral FLICE‐inhibitory protein (vFLIP), encoded by KSHV ORF K13, inhibits KSHV lytic reactivation, implying that the competing endogenous RNA (ceRNA) networks regulated by vFLIP can be modulated to induce the lytic reactivation of latent KSHV, a promising strategy for KSHV‐associated malignancies. Here, we performed whole‐transcriptome sequencing to reveal the global landscape of noncoding RNAs and messenger RNAs (mRNAs) in iSLK‐RGB‐BAC16 cells and iSLK‐RGB‐K13 mutant cells. It showed that vFLIP regulated 227 differentially expressed (DE) long non‐coding RNAs (lncRNAs), 57 DE circular RNAs (circRNAs), 20 DE microRNAs (miRNAs), and 1371 DE mRNAs. Enrichment analysis verified that riboflavin metabolism was simultaneously enriched in DE genes related to miRNAs, lncRNAs, and circRNAs. The upregulated hsa‐miR‐378i and hsa‐miR‐3654, and downregulated miR‐4467, miR‐3163, miR‐4451, and miR‐4257 were significantly enriched in the ceRNA complex network, which contained 9 upregulated and 7 downregulated circRNAs, 5 upregulated and 85 downregulated lncRNAs, 5 upregulated and 35 downregulated mRNAs. Finally, we constructed and validated two vFLIP‐regulated ceRNA networks: circRNA hsa_circ_0070049/hsa‐miR‐378i/SPEG/FOXQ1 and lncRNA AL031123.1/hsa‐miR‐378i/SPEG/FOXQ1. Taken together, the two ceRNA networks may mediate KSHV reactivation. These novel findings refreshed the present understanding of ceRNA network in KSHV lytic induction and provided potential therapeutic targets for KSHV‐associated malignancies.

目的 分析SH3域结合谷氨酸富含蛋白(SH3BGR)基因在宫颈癌组织中的表达及其临床意义,研究SH3BGR蛋白对宫颈癌细胞系HeLa恶性转化、增殖和迁移能力的影响,并初步探索其调控机制.方法 采用免疫组织化学染色方法(IHC)检测SH3BGR蛋白在宫颈癌组织中的表达.运用TCGA数据库和生物信息学相关方法分析SH3BGR表达水平与宫颈癌临床分期和患者生存率的关系.采用PCR进行SH3BGR基因的扩增,将其克隆至pHAGE-CMV-MCS-IzsGreen载体,构建重组慢病毒表达质粒pHAGE-SH3BGR.将重组质粒pHAGE-SH3BGR与包膜质粒pMD2.G和包装质粒psPAX2共转染入人胚肾上皮细胞HEK293T,包装重组慢病毒,运用梯度稀释法测定病毒滴度.将重组慢病毒SH3BGR感染人宫颈癌细胞系HeLa,通过Western blot验证SH3BGR蛋白的表达.采用细胞增殖实验(CCK-8)、细胞划痕实验和软琼脂克隆形成实验分别评价过表达SH3BGR对He-La细胞增殖、迁移及恶性转化能力的影响.运用小干扰RNA(siRNA)敲低人乳头瘤病毒(HPV)E6/E7基因,观察SH3BGR蛋白的表达.结果 SH3BGR蛋白在宫颈癌组织中表达明显高于癌旁组织,SH3BGR高表达宫颈癌患者生存期显著短于低表达患者.慢病毒载体介导的SH3BGR蛋白的过表达可显著提高HeLa细胞的增殖、迁移和恶性转化能力.敲低HPV E6/E7可以抑制SH3BGR蛋白的水平.结论 HPV可能通过E6/E7上调SH3BGR蛋白的表达,促进宫颈癌恶性转化、增殖、迁移,SH3BGR蛋白的高表达不利于宫颈癌患者的生存.

为探讨Sin3A相关蛋白18(SAP18)在宫颈癌组织中的表达情况,并研究SAP18对宫颈癌细胞HeLa增殖和迁移能力的影响与分子机制.利用TCGA和GTEx数据库分析SAP18在宫颈癌患者癌组织和癌旁组织中的表达水平,并计算SAP18表达水平与患者生存率的关系.免疫组织化学染色试验检测宫颈癌组织标本中SAP18蛋白的表达情况.CCK-8试验和软琼脂克隆形成试验检测SAP18对HeLa细胞增殖的影响,细胞划痕试验检测SAP18对HeLa细胞迁移能力的影响.免疫印迹法验证敲低人乳头瘤病毒(HPV)编码的E6、E7蛋白对SAP18表达水平的影响.结果表明:SAP18在宫颈癌组织中呈高表达;SAP18表达水平与宫颈癌患者生存率呈负相关.过表达SAP18可增强HeLa细胞克隆形成、增殖和迁移能力.敲低HPVE6和E7蛋白导致SAP18表达水平下调.这一研究说明HPV通过E6和E7蛋白上调SAP18的表达促进宫颈癌恶性转化、增殖和迁移,SAP18蛋白的高表达不利于宫颈癌患者的生存.

Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs that can decoy other RNAs to inhibit their functions. Kaposi's sarcoma (KS), caused by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), is a highly angiogenic and invasive vascular tumor of endothelial origin commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) induces cell invasion, angiogenesis and cellular transformation; however, the role of circRNAs is largely unknown in the context of KSHV vIRF1. Herein, transcriptome analysis identified 22 differentially expressed cellular circRNAs regulated by vIRF1 in an endothelial cell line. Among them, circARFGEF1 was the highest upregulated circRNA. Mechanistically, vIRF1 induced circARFGEF1 transcription by binding to transcription factor lymphoid enhancer binding factor 1 (Lef1). Importantly, upregulation of circARFGEF1 was required for vIRF1-induced cell motility, proliferation and in vivo angiogenesis. circARFGEF1 functioned as a competing endogenous RNAs (ceRNAs) by binding to and inducing degradation of miR-125a-3p. Mass spectrometry analysis demonstrated that glutaredoxin 3 (GLRX3) was a direct target of miR-125a-3p. Knockdown of GLRX3 impaired cell motility, proliferation and angiogenesis induced by vIRF1. Taken together, vIRF1 transcriptionally activates circARFGEF1, potentially by binding to Lef1, to promote cell oncogenic phenotypes via inhibiting miR-125a-3p and inducing GLRX3. These findings define a novel mechanism responsible for vIRF1-induced oncogenesis and establish the scientific basis for targeting these molecules for treating KSHV-associated cancers.