Shengping Wu

Background: The ORF3 protein of swine hepatitis E virus (HEV-4) is a key virulence factor involved in viral assembly, egress, and host signaling regulation. The mammalian target of rapamycin (mTOR) pathway plays a pivotal role in autophagy, metabolism, and immunity, and is often modulated by viruses to promote replication. However, it remains unknown whether HEV-4 ORF3 modulates the mTOR pathway via circular RNAs (circRNAs). Methods: Using an adenovirus-mediated ORF3 overexpression system in HepG2 cells, we integrated circRNA and transcriptome high-throughput sequencing data, followed by KEGG enrichment analysis to identify mTOR-associated differentially expressed genes. A circRNA–miRNA regulatory network was constructed using bioinformatics tools, and the expression changes of m6A-related genes, including YTHDF3, were evaluated. Results: ORF3 overexpression significantly activated the mTOR pathway (p < 0.05) and led to the identification of 20 mTOR-related circRNAs (e.g., circRNA5142). These circRNAs regulated downstream autophagy and lipid metabolism genes by sponging miRNAs such as hsa-let-7d-5p and hsa-miR-132-3p. Altered YTHDF3 expression indicated possible m6A-dependent epitranscriptomic regulation of the mTOR pathway. Conclusions: Our integrated analysis suggests that HEV-4 ORF3 may modulate the mTOR pathway through a circRNA–miRNA network, perturbing host autophagy and metabolic balance, which may contribute to viral immune evasion. Targeting the ORF3-mediated circRNA-mTOR regulatory axis represents a promising therapeutic approach and provides a theoretical basis for novel anti-HEV-4 strategies.

(1) Background: Swine hepatitis E (SHE) is an emerging zoonotic disease caused by the swine hepatitis E virus (SHEV). The open reading frame 3 (ORF3) protein is a recognized virulence factor of SHEV. Jaundice, the typical clinical sign of SHE, primarily results from disruptions in bile production, secretion, and excretion. However, the mechanism by which SHEV ORF3 influences bile metabolism remains unclear. (2) Methods: Building on our previous work involving adenovirus-mediated overexpression of genotype IV SHEV ORF3 in HepG2 cells and subsequent high-throughput lncRNA/transcriptome sequencing, this study performed KEGG enrichment analysis on differentially expressed lncRNAs. Candidate lncRNAs were validated via qRT-PCR. Cis-regulated target genes were predicted by integrating differentially expressed mRNA data. Furthermore, AlphaFold 3.0 was employed to analyze the molecular binding sites between lncRNA UBC (MSTRG.6881.4) and its target, UBC protein. (3) Results: We identified three lncRNAs associated with the bile secretion pathway (ko 04976) in HepG2 cells expressing genotype IV SHEV ORF3, which were further confirmed by qRT-PCR: lncRNA UBC (MSTRG.6881.4), lncRNA UBC (MSTRG.6881.9), and lncRNA UBC (MSTRG.6881.12). Bioinformatics prediction suggested six lncRNA-mRNA regulatory networks involved these lncRNAs and two downregulated UBC mRNA transcripts (ENST00000540700 and ENST00000536769). Molecular docking indicated that nucleotides 395U and 41C of lncRNA UBC (MSTRG.6881.4) could potentially bind to residues 82Lys, 88Thr, and 90Thr of the UBC protein, with predicted binding energies ranging from −4.73 to −0.75 kcal/mol. (4) Conclusions: The successful identification of bile secretion-related lncRNAs, coupled with the prediction of their regulatory networks and molecular interaction sites, has advanced our understanding of SHEV ORF3 function and the pathogenesis of SHEV infection.

The Swine hepatitis E virus (SHEV) ORF3 protein is pivotal in pathogenesis, yet its regulation of host metabolic homeostasis via endogenous RNA networks remains unclear. This study aimed to elucidate how the SHEV ORF3-mediated circRNA-miRNA network modulates riboflavin metabolism and triggers the aberrant activation of the ko05212 pathway, while also evaluating their physical interactions using AlphaFold 3 structural simulations. To achieve this, high-throughput RNA sequencing, KEGG pathway analysis, and AlphaFold 3 structural simulations were employed to elucidate the circRNA-miRNA-mRNA regulatory network and potential physical interactions. Transcriptomics revealed a “dual activation” of Riboflavin metabolism and Pancreatic cancer pathways. Specifically, we identified an “ENPP Isozyme Switch,” where upregulated hsa_circ_0077855 sponges miR-181a-2-3p, relieving repression of the metabolic enzyme ENPP3 and proto-oncogene KRAS. Furthermore, AlphaFold 3 simulations yielded an extremely low interface predicted Template Modeling score (ipTM = 0.08), refuting direct physical binding, and ORF3 was found to suppress the m6A eraser FTO, suggesting host epigenetic instability. Consequently, SHEV ORF3 induces metabolic remodeling through a dual “epigenetic-post-transcriptional” mechanism: disrupting m6A homeostasis via FTO suppression and constructing a pathogenic ceRNA network via the ENPP3/miR-181a/KRAS axis. These findings highlight the critical role of non-coding RNAs in driving the virus-induced “pre-pathological state”.

(1) Background: Swine hepatitis E (SHE) is a novel zoonotic disease caused by the swine hepatitis E virus. Open Reading Frame 3 (ORF3) is an important virulence protein of swine hepatitis E virus, which promotes the survival and replication of the virus within host cells by regulating the ERK pathway, modulating the transport of growth factors that affect apoptosis, and facilitating the transmission of death signals during HEV infection. (2) Methods: In our previous study, we used adenovirus-mediated overexpression of HEV-4 in swine ORF3 in HepG2 cells, extracted total RNA, and performed high-throughput lncRNA, circRNA, and transcriptome sequencing. Based on the role of ORF3 in regulating the innate immunity of host cells, maintaining ERK activity, inhibiting the transport of growth factors, and suppressing the immune response, the pathways and differentially expressed genes were screened. A clustering analysis was conducted on circRNAs and lncRNAs, which were significantly differentially expressed across the four pathways, and a regulatory network of circRNA-miRNA and lncRNA-mRNA was constructed. (3) Results: In this study, a total of 24 circRNAs and 4 lncRNAs were screened in HepG2 cells expressing ORF3 of HEV-4 in swine, including 8 circRNAs that regulate host cell innate immunity and 18 circRNAs that maintain ERK activity. Four circRNAs inhibited growth factor transport, and three circRNAs inhibited immune responses, predicting the regulatory networks of circRNA-miRNA and lncRNA-mRNA, respectively. (4) Conclusions: In this study, differential genes involved in regulating host cell innate immunity, maintaining ERK activity, inhibiting growth factor transport, and inhibiting immune response processes were successfully screened in HepG2 cells expressing ORF3 in HEV-4 in swine, and the regulatory networks of circRNA-miRNA and lncRNA-mRNA were predicted, respectively, which laid a foundation for further elucidating the function of swine hepatitis E virus ORF3 and elucidating the infection mechanism of swine hepatitis E virus.

The open reading frame 3 (ORF3) protein of the swine hepatitis E virus (SHEV) is a critical virulence factor implicated in viral infection, yet its precise mechanisms remain poorly understood. Circular RNAs (circRNAs) have emerged as key regulators of gene expression during viral infections by functioning as miRNA sponges. This study aimed to identify key circRNAs and construct a potential circRNA-miRNA-mRNA regulatory network associated with the viral infection pathway in HepG2 cells expressing genotype IV SHEV ORF3. Based on our previous high-throughput circRNA and transcriptome sequencing data from HepG2 cells with adenovirus-mediated ORF3 overexpression, we screened for differentially expressed circRNAs and mRNAs linked to viral infection pathways. Using bioinformatic tools, we predicted miRNAs targeted by these mRNAs and those that could bind to the circRNAs, ultimately constructing a competing endogenous RNA (ceRNA) network with Cytoscape. We identified 31 differentially expressed circRNAs and 7 mRNAs (HSPA8, HSPA1B, EGR2, CXCR4, SOCS3, NOTCH3, and ZNF527) related to viral infection. A potential ceRNA network comprising 32 circRNAs, 23 miRNAs, and the 7 mRNAs was constructed. Core circRNAs, including ciRNA203, circRNA14936, and circRNA5562, may act as miRNA sponges to regulate the expression of these mRNAs. This network suggests a novel mechanism by which SHEV ORF3 might modulate host cell functions to facilitate viral infection.

Brucellosis is a severe zoonotic disease caused by Brucella infection, which remains prevalent in several regions worldwide and poses a significant public health challenge. The host deploys complex immune mechanisms to combat the pathogen, including the recognition of pathogenic signals, secretion of inflammatory factors, and activation of innate and adaptive immune responses. Brucella, as a facultative intracellular pathogen, replicates within host cells and establishes chronic infections through diverse immune evasion strategies. These include subversion of Toll-like receptor (TLR) signaling, disruption of antigen presentation, and interference with T cell responses. This review focuses on Brucella species with significant human infectivity, such as B. melitensis and B. abortus, summarizing their interactions with the host immune system. Recent studies have highlighted TLR pathway inhibition, antigen presentation impairment, and T cell dysregulation as key mechanisms of immune evasion. Understanding these processes is crucial for elucidating Brucella pathogenesis and developing novel therapeutic and vaccine strategies against brucellosis.

(1) Background: Hepatitis E (HE) is a novel zoonotic disease caused by hepatitis E virus (HEV). In particular, swine hepatitis E virus (SHEV) genotype IV is one of the main genotypes that infect humans. Open reading frame 3 (ORF3) is an important virulence protein of SHEV, which is involved in virus assembly, release, and regulation of host cell signaling pathways. Circular RNAs (circRNAs), as a type of competitive endogenous RNA (ceRNA), have a closed-loop structure and are special non-coding RNA molecules. They participates in the regulation of multiple biological processes by adsorbing microRNAs (miRNAs). Riboflavin, also known as vitamin B2, is a component of the coenzyme of flavoenzymes in the body. When there is a deficiency of riboflavin, it will affect the biological oxidation process of the host, leading to metabolic disorders. In addition, riboflavin can also affect the synthesis, transportation and decomposition of lipids in the body. It mainly maintains the normal transportation process of fat in the liver. Therefore, the deficiency of riboflavin will lead to the disorder of lipid metabolism in the body. Thus, viral hepatitis is closely related to riboflavin metabolism. However, there are very few reports on SHEV ORF3 affecting the riboflavin metabolism of target cells and thereby influencing viral infection. Therefore, this study investigates this highly significant scientific issue. (2) Methods: In the previous research of our group, adenovirus was used to mediate the overexpression of SHEV ORF3 genotype IV in HepG2 cells. Total RNA was extracted for high-throughput sequencing of circRNAs and transcriptome. KEGG functional enrichment analysis was performed on the data to identify the differentially expressed circRNAs and miRNAs after SHEV infection, and the relevant circRNA-miRNA network in the riboflavin metabolism pathway in HepG2 cells was found. (3) Results: We identified 4 circRNAs in the riboflavin metabolism pathway of HepG2 cells expressing the ORF3 protein of SHEV genotype IV and successfully found 26 relevant circRNA-miRNA networks. (4) Conclusion: We successfully screened and identified circRNAs related to riboflavin metabolism, further identifying the circRNA-miRNA network and its functional targets. For the first time, we investigated the key mechanism by which ORF3 protein influences riboflavin metabolic pathways in target cells through circRNAs, preliminarily revealing that ariboflavinosis can lead to lipid metabolic disorder in the organism. This indicates a close association between viral HE and riboflavin metabolism.

Hepatitis E virus (HEV) is one of the pathogens that cause viral hepatitis, and its clinical symptoms can manifest as acute, chronic viral hepatitis, or asymptomatic infection. Among them, swines are the main animal source of HEV. Open reading frame 3 (ORF3) is a multifunctional protein essential for swine hepatitis E virus (SHEV) infection and release, involved in biological processes such as intracellular signal transduction regulation. In our preliminary research, we utilized adenovirus-mediated overexpression of type IV SHEV ORF3 in HepG2 cells, extracted total RNA, and performed high-throughput long non coding RNAs (lncRNAs) and transcriptome sequencing. In this study, we screened and analyzed lncRNAs involved in the GO pathway: viral process (GO: 0016032), and combined them with differentially expressed mRNAs for target gene prediction. We identified two lncRNAs—lncRNA AL137002 (MSTRG. 7478) and lncRNA AL049840 (MSTRG. 8427)—that are associated with viral progression and have p ≤ 0.05 in HepG2 cells expressing ORF3 of porcine hepatitis E virus type IV. We predicted their five lncRNA-mRNA networks, which are lncRNA AL137002 (MSTRG. 7478)-ENST0000375440, lncRNA AL137002 (MSTRG. 7478)-ENST0000375441, lncRNA AL049840(MSTRG. 8427)-ENST0000246489, lncRNA AL049840 (MSTRG. 8427)-ENST0000554280 and lncRNA AL049840 (MSTRG. 8427)-ENST0000452929, and were used to predict their lncRNA mRNA binding sites and construct relevant molecular models. This will lay a solid foundation for further revealing the function of SHEV ORF3 and elucidating the mechanism of SHEV infection.

Background: Swine hepatitis E (HEV) is a zoonotic infectious disease caused by the swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key virulence factor in swine HEV, playing a crucial role in the release of viral particles, the modulation of the host innate immune response, and regulation of autophagy and apoptosis, etc. However, its main function and pathogenic mechanism remain incompletely understood. Results: In our study, adenoviruses ADV4-ORF3 and ADV4-GFP were successfully constructed and mediated the overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP in HepG2 cells. A total of 217 differentially expressed messenger RNAs (mRNAs) were screened by high-throughput sequencing, and 27 statistically significant differentially expressed genes were screened for further quantitative real-time reverse transcription (qRT-PCR) verification by functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]). They are mainly involved in six pathways: the cellular response to unfolded protein, inflammatory response, cytokine activity, TNF signaling pathway, influenza A, and pathways in cancer. In a comparative analysis of transcriptome and mRNA expression profiles of lncRNA sequencing, the results showed that 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified. Conclusions: Eight differential genes, HSPA1A, HSPA1B, PLD3, RELA, GPI, SAMHD1, RPS6KA4, and PIK3CB, were successfully verified. Comparing and analyzing the results of the two sequencing methods indicated that the 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified in SHEV ORF3-expressing HepG2 cells, which has laid a genetic foundation for the physiological function and mechanism of SHEV ORF3.

Hepatitis E (Hepatitis E, HE) is an acute and chronic infectious hepatitis caused by hepatitis E virus (Hepatitis E Virus, HEV) infection, which is responsible for most acute hepatitis in the world and is a significant public health problem. The pathogen, HEV, has three Open Reading Frames (ORFs) ORF1, ORF2, and ORF3, each of which has a different function. Most of the current research is focused on ORF1 and ORF2, while the research on ORF3 is still relatively small. To provide more ideas for the study of HEV pathogenesis and the prevention and treatment of HE, this paper reviews the effects of ORF3 on the ERK pathway, growth factors, immune response, and virus release.