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The NF-κB pathway is an essential signalling cascade in the defence against viral infections, including African swine fever virus (ASFV) infection. ASFV encodes more than 151 proteins via its own transcription machinery and possesses a great capacity to evade or subvert antiviral innate immune responses. Although some of these viral proteins have been reported, many remain unknown. Here, we show that pD345L, an ASFV-encoded lambda-like exonuclease, acts as an inhibitor of cGAS/STING-mediated NF-κB signalling by blocking the IkappaB kinase (IKKα/β) activity. Specifically, we showed that overexpression of pD345L suppresses cGAS/STING-induced IFNβ and NF-κB activation, resulting in decreased transcription of IFNβ and several proinflammatory cytokines, including IL-1α, IL-6, IL-8, and TNFα. In addition, we showed that pD345L acts at or downstream of IKK and upstream of p65. Importantly, we found that pD345L associates with the KD and HLH domains of IKKα and the LZ domain of IKKβ and thus interrupts their kinase activity towards the downstream substrate IκBα. Finally, we showed that pD345L-mediated inhibition of NF-κB signalling was independent of its exonuclease activity. Considering these results collectively, we concluded that pD345L blocks IKKα/β kinase activity via protein–protein interactions and thus disrupts cGAS/STING-mediated NF-κB signalling.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky’s disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS–STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS–STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS–STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3.

The p53 gene is mutated in more than 50% of human tumors. Mutant p53 exerts an oncogenic function and is often highly expressed in cancer cells due to evasion of proteasome-dependent degradation. Thus, reactivating proteasome-dependent degradation of mutant p53 protein is an attractive strategy for cancer management. Previously, we found that arsenic trioxide (ATO), a drug for acute promyelocytic leukemia, degrades mutant p53 protein through a proteasome pathway. However, it remains unclear what is the E3 ligase that targets mutant p53 for degradation. In current study, we sought to identify an E3 ligase necessary for ATO-mediated degradation of mutant p53. We found that ATO induces expression of Pirh2 E3 ligase at the transcriptional level. We also found that knockdown of Pirh2 inhibits, whereas ectopic expression of Pirh2 enhances, ATO-induced degradation of mutant p53 protein. Furthermore, we found that Pirh2 E3 ligase physically interacts with and targets mutant p53 for polyubiquitination and subsequently proteasomal degradation. Interestingly, we found that ATO cooperates with HSP90 or HDAC inhibitor to promote mutant p53 degradation and growth suppression in tumor cells. Together, these data suggest that ATO promotes mutant p53 degradation in part via induction of the Pirh2-dependent proteasome pathway.

Marek’s disease virus (MDV), an alphaherpesvirus, causes severe immunosuppression and T cell lymphomas in chickens, known as Marek’s disease (MD), an economically important poultry disease primarily controlled by vaccination. Importantly, it also serves as a comparative model for studying herpesvirus-induced tumor formation in humans. MDV encodes more than 100 genes, most of which have unknown functions. MDV LORF1 is unique to serotype I MDV (MDV-1), lacking homologs in other herpesviruses, and has not been explored yet. To this end, an infectious bacterial artificial chromosome (BAC) harboring the complete genome of the MDV-1 very virulent strain Md5 was generated, and the rescued rMd5 maintained biological properties similar to the parental virus both in vitro and in vivo . Subsequently, rMd5ΔLORF1, a recombinant Md5 virus deficient in pLORF1 expression, was generated by a frameshift mutation in the LORF1 gene. Chickens infected with rMd5ΔLORF1 exhibited a lower mortality rate and delayed bursal atrophy than those infected with the parental rMd5 and the revertant virus (rMd5-reLORF1). Consistently, viral loads of rMd5ΔLORF1 were obviously lower than those of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen. Importantly, we found that pLORF1 deficiency impairs viral replication in bursal B cells. Furthermore, we showed that pLORF1 associated with the cellular membrane, interacted with MDV structural proteins, and exhibited punctate colocalization with tegument or capsid proteins in the cytoplasm. Taken together, this study demonstrates for the first time that the MDV-1 unique gene LORF1 is involved in MDV-induced bursal atrophy but not in tumor formation.

Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus causing acute diarrhea and high mortality in neonatal suckling piglets, resulting in huge economic losses for the global swine industry. The replication, assembly and cell egression of PEDV, an enveloped RNA virus, are mediated via altered intracellular trafficking. The underlying mechanisms of PEDV secretion are poorly understood. In this study, we found that the histone deacetylase (HDAC)-specific inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), facilitate the secretion of infectious PEDV particles without interfering with its assembly. We found that PEDV N protein and its replicative intermediate dsRNA colocalize with coat protein complex II (COPII)-coated vesicles. We also showed that the colocalization of PEDV and COPII is enhanced by the HDAC-specific inhibitors. In addition, ultrastructural analysis revealed that the HDAC-specific inhibitors promote COPII-coated vesicles carrying PEDV virions and the secretion of COPII-coated vesicles. Consistently, HDAC-specific inhibitors-induced PEDV particle secretion was abolished by Sec24B knockdown, implying that the HDAC-specific inhibitors-mediated COPII-coated vesicles are required for PEDV secretion. Taken together, our findings provide initial evidence suggesting that PEDV virions can assemble in the endoplasmic reticulum (ER) and bud off from the ER in the COPII-coated vesicles. HDAC-specific inhibitors promote PEDV release by hijacking the COPII-coated vesicles.

P73, a member of the p53 family, plays a critical role in neural development and tumorigenesis. Due to the usage of two different promoters, p73 is expressed as two major isoforms, TAp73 and ΔNp73, often with opposing functions. Here, we reported that transcriptional factor DEC1, a target of the p53 family, exerts a distinct control of TAp73 and ΔNp73 expression. In particular, we showed that DEC1 was able to increase TAp73 expression via transcriptional activation of the TAp73 promoter. By contrast, Np73 transcription was inhibited by DEC1 via transcriptional repression of the ΔNp73 promoter. To further explore the underlying mechanism, we showed that DEC1 was unable to increase TAp73 expression in the absence of HDAC8, suggesting that HDAC8 is required for DEC1 to enhance TAp73 expression. Furthermore, we found that DEC1 was able to interact with HDAC8 and recruit HDAC8 to the TAp73, but not the ΔNp73, promoter. Together, our data provide evidence that DEC1 and HDAC8 in differentially regulate TAp73 and ΔNp73 expression, suggesting that this regulation may lay a foundation for a therapeutic strategy to enhance the chemosensitivity of tumor cells.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are major sensors against viral infection, but their roles in DNA virus infection largely remain unknown. This study found that a previously uncharacterised protein, pS183L, negatively regulates RLR signalling by suppressing MDA5 oligomerisation. Specifically, we showed that the overexpression of pS183L suppresses MDA5 but not cGAS-STING or RIG-I-induced IFN-β activation. Consistently, pS183L inhibited high molecular weight poly (I:C) activated IFN-β production. Furthermore, we demonstrated that pS183L interacts with CARDs and the MDA5 Helicase domain, consequently blocking MDA5 oligomerisation and the MDA5-MAVS interaction. Taken together, we concluded that pS183L blocks MDA5 oligomerisation through protein–protein interaction and thus disrupts MDA5-mediated IFN-β signalling.

African swine fever virus (ASFV) causes highly contagious swine disease, African swine fever (ASF), thereby posing a severe socioeconomic threat to the global pig industry and underscoring that effective antiviral therapies are urgently required. To identify safe and efficient anti-ASFV compounds, a natural compound library was screened by performing an established cell-based ELISA in an ASFV-infected porcine alveolar macrophage (PAM) model. In total, 6 effective anti-ASFV compounds with low cytotoxicity were identified. Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid, was the most potent inhibitor effect with an IC of 0.3223 μM. To further investigate the mechanism through which CEP inhibits ASFV replication, transcriptome profiles were generated in PAMs treated with CEP and/or infected with ASFV. ASFV infection dramatically altered immune response-associated gene expression. CEP treatment upregulated the expression of cholesterol biosynthesis-related genes, regardless of infection status. According to time-of-addition experiments, CEP primarily exerts its antiviral effect during the early stages of ASFV infection, specifically by inhibiting viral entry. Transcriptomic analysis suggested that CEP blocks ASFV entry through the clathrin-mediated endocytosis pathway by increasing EHD2 gene expression in macrophages. Disrupting EHD2 with small interfering RNA promoted ASFV entry into clathrin-positive vesicles. Finally, the protective effect of CEP was evaluated using ASFV-infected pigs. CEP could provide partial protection against ASFV infection, as indicated by an increase in survival time from 9.67 days to 16.67 days. Our findings imply that CEP exhibits potential antiviral activity against ASFV infection in PAMs, positioning it as a promising therapeutic strategy for ASF.

DNA damage response (DDR) is an evolutionarily conserved mechanism by which eukaryotic cells sense DNA lesions caused by intrinsic and extrinsic stimuli, including virus infection. Although interactions between DNA viruses and DDR have been extensively studied, how RNA viruses, especially coronaviruses, regulate DDR remains unknown. A previous study showed that the porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the Coronaviridae family, induces DDR in infected cells. However, the underlying mechanism was unclear. This study showed that PEDV activates the ATM-Chk2 signaling, while inhibition of ATM or Chk2 dampens the early stage of PEDV infection. Additionally, we found that PEDV-activated ATM signaling correlates with intracellular ROS production. Interestingly, we showed that, unlike the typical γH2AX foci, PEDV infection leads to a unique γH2AX staining pattern, including phase I (nuclear ring staining), II (pan-nuclear staining), and III (co-staining with apoptotic bodies), which highly resembles the apoptosis process. Furthermore, we demonstrated that PEDV-induced H2AX phosphorylation depends on the activation of caspase-7 and caspase-activated DNAse (CAD), but not ATM-Chk2. Finally, we showed that the knockdown of H2AX attenuates PEDV replication. Taken together, we conclude that PEDV induces DDR through the ROS-ATM and caspase7-CAD-γH2AX signaling pathways to foster its early replication.

Bovine gammaherpesvirus 4 (BoHV-4) is a common virus detected in bovine with respiratory disease worldwide. In this study, we identified and characterized a novel BoHV-4 strain, referred as HB-ZJK, in vaginal swabs collected from cattle in China, 2022. The long unique region (LUR) of HB-ZJK is 10,9811 bp in length. It shares 99.17% to 99.38% nucleotide identity to five BoHV-4 strains available in GenBank and the highest similarity was seen with BoHV-4V. test (JN133502.1) strain (99.38%). Mutations, insertions or deletions were observed mainly in HB-ZJK gB (ORF8), TK (ORF21), gH (ORF22), MCP (ORF25), PK (ORF36), gM (ORF39), and gL (ORF47) genes compared to its genomic coordinates. Phylogenetic analyses of gB and TK genes showed that HB-ZJK clustered with China 512 (2019), B6010 (2009), and J4034 (2009) strains, demonstrating that the isolated HB-ZJK belongs to genotype 1. This is the first report that has revealed a comprehensive genome profile of BoHV-4 strain in China. This study will provide foundation for epidemiological investigations of BoHV-4 and contribute to the molecular and pathogenic studies of BoHV-4.