A rapid, specific, and sensitive quantitative PCR assay was developed for the detection of Porcine Teschovirus (PTV). Specific primers were designed on the basis of conserved regions of complete PTV genome sequences retrieved from GenBank, and a recombinant plasmid was constructed to generate a standard curve. Reaction conditions were optimized, and the sensitivity, specificity, and reproducibility of the assay were systematically evaluated. The detection limit of the Reverse Transcription Quantitative PCR(RT-qPCR) assay was 1 & times;101 copies/mu L, which was approximately 100-fold more sensitive than that of conventional PCR (1 & times;103 copies/mu L). A single melting curve peak confirmed the specificity of the assay, and no cross-reactivity was observed with other common porcine viruses. The coefficient of variation was less than 1.1%, indicating excellent reproducibility. Application of the established method to 541 clinical samples revealed a positive rate of 68.4%, consistent with conventional PCR results. These results demonstrate that the developed RT-qPCR assay is a reliable tool for the detection, surveillance, and epidemiological investigation of PTV.

Peptidyl-prolyl isomerase B (PPIB), a member of the peptidyl-prolyl cis-trans isomerase family, is well-documented to facilitate viral propagation by interactions with viral proteins. However, its regulatory roles in the replication of bovine ephemeral fever virus (BEFV) or vesicular stomatitis virus (VSV), as well as in host innate immune responses remain unexplored. In this study, we demonstrate that PPIB enhances the replication of BEFV and VSV and suppresses the host type I interferon (IFN-I) response. Mechanistically, PPIB interacts with phenazine biosynthesis-like domain-containing protein (PBLD), a positive regulator of innate immunity, and triggers its degradation via the ubiquitin-proteasome pathway. Further analyses showed that PPIB enhances the interaction between the E3 ubiquitin ligase March2 and PBLD, thereby facilitating PBLD ubiquitination and subsequent degradation. This PPIB-mediated, March2-dependent degradation of PBLD inhibits IFN-I production, ultimately enhancing viral replication. Collectively, our findings unveil an unrecognized role of PPIB in regulating IFN-I responses and viral replication through the PPIB-March2-PBLD signaling axis, providing novel insights for the development of broad-spectrum antiviral therapeutics.

The activation of type I interferon (IFN-I) signaling is crucial for defending host cells against viral infections. A comprehensive IFN-I response necessitates the activation of several cellular factors, among them Interferon Regulator Factor 7 (IRF7). Nonetheless, the mechanisms governing IRF7 inactivation in response to viral infection remain largely unknown. Here, we illustrate that Cluster of differentiation 97 (CD97), a G protein-coupled receptor, interacts with PPM1G via intracellular Arg-819 and Arg-822 residues. PPM1G then recruits and dephosphorylates IRF7, leading to its inhibition. CD97-mediated inactivation of IRF7 impedes its translocation into the nucleus and subsequent activation of IFN-I, ultimately promoting the viral replication. Moreover, mice lacking CD97 display heightened resistance to viral infection. The compound sanguinarine (SANG) hinders viral replication by dampening CD97 expression. This study provides a basis for CD97 as a potential antiviral target and SANG as a candidate antiviral small molecule drug.

PRKC Apoptosis WT1 Regulator (PAWR) has been implicated in tumorigenesis. However, its role in antiviral innate immunity remains unexplored. Here, we demonstrate that PAWR transcriptionally upregulates RIG-I expression through the transcription factor X-box binding protein 1 (XBP1). Mechanistically, PAWR potentiates the ATF6/IRE1-XBP1 pathway to upregulate spliced form XBP1 (XBP1s) and simultaneously promotes PIM2-mediated phosphorylation of XBP1s at Ser68. Activated XBP1s binds the RIG-I promoter, driving RIG-I expression and amplifying IFN-I responses during RNA virus infection. Most intriguingly, PAWR-silenced THP-1 cells and primary macrophages exhibit attenuated anti-RNA viral IFN-I responses. Pawr-deficient mice are more susceptible to RNA virus challenge. Notably, Arylquin 1, the small molecule activator of PAWR, inhibits VSV replication by boosting the RIG-I-mediated IFN-I response in a PAWR-dependent manner. Collectively, our findings highlight a critical function of PAWR in antiviral innate immunity via the PIM2-XBP1s-RIG-I signaling axis, establishing its potential as a promising therapeutic target for RNA viral infections.

Sec8, an exocyst complex subunit, is pivotal in facilitating the docking of exocytic vesicles to fusion sites on the plasma membrane. However, its involvement in the antiviral innate immune response and virus replication remains unclear. In this study, Sec8 is identified as a novel positive regulator of RIG-I, enhancing the IFN-I signaling response against RNA viruses both in vivo and in vitro. Additionally, Sec8 stabilizes RIG-I by inhibiting its ubiquitination and subsequent proteasome-mediated degradation. Mechanistically, STUB1 degrades RIG-I via K48-linked ubiquitination at Lys190, while Sec8 suppresses STUB1 mRNA by reducing the expression of p53 and competes with STUB1 for binding to RIG-I’s CARD domain, thereby preventing STUB1-mediated RIG-I degradation. Importantly, Sec8-deficient mice were more susceptible to RNA virus infection compared to wild-type mice. These findings elucidate a mechanism that Sec8 positively regulates RIG-I in the antiviral innate immune response, offering insights for developing novel therapeutic strategies and targeted antiviral medications.

SEC10 (EXOC5), a central subunit of the exocyst complex, plays a critical role in vesicle trafficking. However, its function in bovine herpesvirus 1 (BoHV-1) triggered-antiviral innate immunity and viral replication remains unclear. In this study, we identify SEC10 as a key negative regulator of antiviral immune responses. SEC10 suppresses the transcriptional expression of JAK1, thereby significantly impairing activation of the JAK-STAT signaling pathway, dampening type I interferon (IFN-I)-mediated antiviral immunity, and promoting BoHV-1 replication. Mechanistically, SEC10 downregulates the expression of the transcription factor KLF15 and subsequently modulates JAK1 transcription in a KLF15-dependent manner, establishing a “SEC10-KLF15-JAK1” regulatory axis. Collectively, our findings not only reveal SEC10 as a novel immunomodulatory factor but also uncover a previously unrecognized KLF15-dependent transcriptional mechanism that fine-tunes innate immune responses. This work provides new insights into the complex regulatory network of the JAK-STAT signaling pathway and identifies a potential therapeutic target for combating BoHV-1 infection.

Phenazine biosynthesis-like domain-containing protein (PBLD) has been proven to be a critical regulator of tumor suppression and antiviral innate immunity; however, its role in pyroptosis remains unexplored. Our current investigation shows that PBLD promotes pyroptosis in bovine parainfluenza virus 3 (BPIV3)- or herpes simplex virus type 1 (HSV-1)-triggered HeLa cells, along with BPIV3- or bovine ephemeral fever virus (BEFV)-infected BHK-21 cells, as manifested by increased hallmark features of pyroptosis, including cell swelling, plasma membrane disintegration, elevated lactate dehydrogenase (LDH) release, and reduced cell survival. Further studies reveal that PBLD facilitates virus-induced pyroptosis mediated by GSDME N-terminal cleavage but independent of GSDMD cleavage. Using caspase-specific inhibitors and knockout cell lines, we identify Caspase-3, but not Caspase-8, as essential for virus-induced GSDME-dependent pyroptosis. Mechanistically, PBLD enhances Caspase-3 activation by upregulating PUMA mRNA levels via the NF-κB signaling pathway. Furthermore, silencing of NF-κB abolishes PBLD-induced PUMA upregulation and Caspase-3 and GSDME cleavage. In summary, these findings reveal that PBLD potentiates virus-triggered pyroptosis through the NF-κB/PUMA/Caspase-3/GSDME signaling pathway. This investigation provides unprecedented understanding of the molecular mechanisms by which PBLD regulates cell death and highlights its promise as a pharmacological target for viral infections and inflammatory diseases.

EXOC5/SEC10, the central subunit of the exocyst complex, is crucial for the trafficking of secretory vesicles to the plasma membrane. However, its role in innate immunity and viral replication remains unclear. Here we demonstrate that EXOC5 acts as a negative regulator of DNA virus-triggered CGAS-STING1 signaling via targeting STING1. Mechanistically, EXOC5 facilitates the autophagic degradation of STING1 via K63-linked polyubiquitination at Lys224 and Lys338 by the E3 ligase TRIM56, which serves as a recognition signal for the cargo receptor SQSTM1/p62 (sequestosome 1). Furthermore, EXOC5 inhibits antiviral innate immunity and promotes viral replication via EXOC5-TRIM56-STING1-SQSTM1 signal transduction. More importantly, myeloid-specific deletion of Exoc5 in mice improves survival and reduces viral load. In general, these findings revealed a negative feedback loop of type I interferon signaling through the EXOC5-TRIM56-STING1-SQSTM1 axis, which has the potential to serve as a new target for the development of antiviral therapeutics that regulate the host immune response.Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; EXOC5/SEC10: exocyst complex component 5; HAdV-4: human adenovirus type 4; HSV-1: herpes simplex virus type 1; HT-DNA: herring testis deoxyribonucleic acid; IFN: interferon; IRF3: interferon regulatory factor 3; ISD: interferon stimulatory DNA; PMs: peritoneal macrophages; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; VACV70: 70-mers of dsDNA representing the genome of vaccinia virus.

Stimulator of interferon response cGAMP interactor (STING), the central transducer of the cGAS-STING signaling axis, governs type I interferon (IFN-I) production that is essential for antiviral innate immunity. Modulating STING activity and stability offers potential therapeutic strategies for viral and autoimmune diseases. Here, we demonstrate that testis-expressed protein 264 (TEX264), an endoplasmic reticulum-selective autophagy (ER-phagy) receptor, shows upregulated expression following Herpes simplex virus 1 (HSV-1) infection. Overexpression of TEX264 inhibits the activation of IFN-I signaling triggered by HSV-1 or poly(dA:dT), and enhances HSV-1 replication. Mechanistically, TEX264 interacts with WIPI2 to induce ER-phagy, leading to the degradation of STING and the negative regulation of the IFN-I response. Our findings position TEX264 as a critical regulator of the innate immune response to DNA viruses.

Acyl-CoA binding domain containing protein 3 (ACBD3) is a Golgi protein implicated in multiple cellular processes. However, its function in negative-strand RNA virus infection and innate immune responses remains poorly understood. Here, we identify ACBD3 as a novel positive regulator of host defense that potently inhibits vesicular stomatitis virus (VSV) replication in HeLa cells with ACBD3 overexpression or knockdown. Mechanistically, our investigation unveils a previously unknown ACBD3-TRIM21-MAVS axis. Co-immunoprecipitation combined with mass spectrometry analysis reveals that ACBD3 interacts with the E3 ubiquitin ligase TRIM21, and this interaction is crucial for stabilizing the mitochondrial antiviral-signaling protein (MAVS). During VSV infection, ACBD3 enhances TRIM21 protein levels, thereby promoting MAVS accumulation and facilitating the activation of type I interferon signaling. Collectively, our findings elucidate a novel mechanism by which ACBD3 sustains innate immunity via TRIM21 to restrict VSV replication, providing a potential therapeutic target and strategy for combating negative-strand RNA viruses.

Sec6 is one of the eight subunits of the exocyst complex, playing a specific role in cell-cell adhesion and vesicle trafficking. However, its role in the replication of bovine ephemeral fever virus (BEFV) and the antiviral innate immune response has remains unclear. In this study, we demonstrate that Sec6 inhibits the BEFV-triggered type I IFN (IFN-I) signaling response and promotes viral replication. Further research revealed that Sec6 degrades mitochondrial antiviral signaling protein (MAVS) through the autophagy pathway. Mechanistically, Sec6 promotes the association between autophagy receptor P62 and MAVS, enhancing autophagy degradation of MAVS. Silencing Sec6 inhibits the interaction between MAVS and P62. In addition, Sec6 failed to degrade MAVS in P62-knockdown cells, resulting in the loss of its ability to suppress IFN-I signaling and enhance viral replication. This study reveals a previously unrecognized role of Sec6 in modulating the antiviral innate immunity and its impact on BEFV replication.

Mitochondrial single-stranded DNA-binding protein 1 (SSBP1), a component of the mitochondrial DNA replisome, is involved in DNA replication, repair and maintenance of mitochondrial DNA. However, its function in the bovine ephemeral fever virus (BEFV) infection has not been described. Herein, we found that SSBP1 acted as an essential negative regulator of mitochondrial antiviral signaling protein (MAVS) to maintain immune homeostasis. Under BEFV infection, SSBP1 was upregulated in host cells, which boosted the susceptibility to virus. SSBP1 dramatically impaired BEFV-triggered antiviral immune response by degrading MAVS. Mechanistically, SSBP1 induced K48-linked ubiquitination of MAVS catalyzed by Smad ubiquitin regulatory factor 1 (Smurf1), thereby promoting ubiquitinated-MAVS for proteasomal degradation. Most importantly, we identified A01, a Smurf1-specific inhibitor that blocked degradation of MAVS induced by SSBP1 and enhanced MAVS-mediated antiviral signaling, consequently suppressing BEFV replication. Taken together, our findings reveal that SSBP1 restricts BEFV-induced innate immune activation through SSBP1-Smurf1-MAVS signaling axis, indicating its potential role as a therapeutic target for viral infections.

The exocyst complex is a heterooctameric protein complex, the individual components of the complex are thought to act on specific biological processes. However, the role of Sec10, the central subunit of the complex, in host defense and viral replication remains unclear. Here, we reported that Sec10 significantly impairs the activation of JAK-STAT signal pathway of type I IFN (IFN-I) response against both DNA- and RNA-viruses, and promotes viral replication, respectively. Mechanistically, Sec10 interacts with E3 ligase STUB1, promotes the interaction of STUB1 and STAT1, and consequently accelerate STUB1-mediated proteasomal degradation of STAT1 via K6-linked polyubiquitination at Lys240 and Lys652, thus weakens STAT1 triggered antiviral immune responses. More importantly, myeloid-specific deletion of Sec10 in mice showed enhanced IFN-I response against viral infection and improved survival of mice. Collectively, these findings demonstrate that Sec10 attenuates the JAK-STAT signaling pathway by targeting STAT1 for proteasomal degradation and identifies a previously unknown function of Sec10 in antiviral innate immunity and viral replication.

Sec6 is a constituent subunit of the exocyst complex, which plays a critical role in exocytosis. However, its involvement in DNA virus infection induced-innate immune responses has remained unclear. Here, we demonstrate that Sec6 suppresses the innate immune response triggered by bovine herpesvirus 1 (BoHV-1) and promotes viral infection. Specifically, Sec6 directly targets the STING for degradation, thereby suppressing STING-dependent innate immune signaling pathways. Mechanistically, Sec6 promotes the interaction between the selective autophagy receptor NDP52 and STING, and facilitates the autophagic degradation of STING. Notably, knockdown of NDP52 abolishes Sec6-mediated suppression of IFN-β and interferon-stimulated genes (ISGs) production, and impairs Sec6's ability to enhance BoHV-1 replication. Collectively, these findings reveal a novel mechanism by which BoHV-1 evades host innate immunity and highlight the Sec6-NDP52-STING axis as a potential target for the development of antiviral therapies.

Bovine parainfluenza virus type 3 (BPIV3) is a major pathogen responsible for bovine respiratory disease syndrome, posing a significant threat to cattle health. Currently, there are no effective antiviral drugs targeting BPIV3 infection. Our previous research identified Cluster of Differentiation 97 (CD97) as a positive regulator of RNA virus replication, and that sanguinarine can inhibit CD97 expression. However, the role of sanguinarine in BPIV3 infection and its underlying mechanisms remained unclear. In this study, we demonstrated that sanguinarine suppresses BPIV3 replication in a dose- and time-dependent manner. This inhibitory effect was significantly attenuated upon CD97 knockout. Furthermore, sanguinarine did not affect the CD97 mRNA level. Additionally, after treatment with the inhibitors Bafilomycin A1 and Z-VAD-FMK, sanguinarine no longer inhibited CD97 protein expression. Mechanistically, sanguinarine promotes CD97 degradation through both the autophagy receptor NBR1 and the apoptotic caspases-3/8, while also enhancing autophagy and apoptosis. Together, these findings reveal the antiviral potential of sanguinarine against BPIV3 and suggest its promise as a candidate therapeutic agent.

Endosome-associated trafficking regulator 1 (ENTR1) is implicated in cell apoptosis, cytokinesis, and adipogenesis, but its role in antiviral innate immunity has not been elucidated. In this study, we identify ENTR1 as a positive regulatory factor for type I interferon (IFN-I) signaling pathway, which suppresses bovine parainfluenza virus type 3 (BPIV3) and vesicular stomatitis virus (VSV) replication. Further investigations revealed that ENTR1 deficiency enhanced Nip3-like protein X (NIX)-mediated mitophagy, leading to accelerated degradation of mitochondrial antiviral signaling protein (MAVS) during viral infection. Mechanistically, ENTR1 knockout resulted in increased accumulation of NIX on mitochondria, which promoted the autophagic degradation of MAVS. Importantly, silencing NIX rescued MAVS protein levels and significantly reduced viral titers in ENTR1-deficient cells. Moreover, NIX silencing prevented the degradation of MAVS and consequently reduced viral titers in ENTR1-deficient cells. Consequently, our findings reveal a novel regulatory axis in which ENTR1 stabilizes MAVS by suppressing NIX-dependent mitophagy, thereby enhancing antiviral IFN-I responses. This study not only uncovers a previously unrecognized function of ENTR1 in antiviral immunity but also identifies ENTR1 as a potential target for developing broad-spectrum antiviral therapeutics against RNA viruses.

IBRV causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses. To improve diagnostic efficiency, this study developed a Nano-qPCR assay targeting the conserved UL50 gene of IBRV. The assay demonstrated high specificity, with no cross-reactivity to other tested bovine pathogens. Sensitivity testing demonstrated that Nano-qPCR could detect IBRV at concentrations as low as 3.5 × 10⁰ copies/μL, whereas conventional qPCR failed to detect it at this level. The assay was validated using clinical samples (nasal/oral swabs, feces, blood, and tissues) from unvaccinated cattle suspected of IBRV infection. Among 68 samples, Nano-qPCR detected six additional positive cases missed by conventional qPCR, which were later confirmed as IBRV-positive by viral isolation in MDBK cells. This confirmed the superior sensitivity of Nano-qPCR. In conclusion, the Nano-qPCR assay offers a rapid, cost-effective, and highly sensitive method for IBRV detection, making it a promising tool for disease surveillance and control in the cattle industry.

Phenazine biosynthesis-like domain-containing protein (PBLD) and Cedrelone have been identified as tumor suppressors. However, their roles in virus infection remain unclear. Here, we demonstrate that PBLD upregulates the type I interferon (IFN-I) response through activating NF-kappaB (NF-κB) signaling pathway to resist viral infection in cells and mice. Mechanistically, PBLD activates NF-κB signaling pathway during viral infection via blocking tripartite motif containing 21 (TRIM21)-mediated phosphorylated inhibitory kappa B kinase beta (IKKβ) degradation. Furthermore, we show Cedrelone inhibits viral replication by increasing the PBLD protein expression and subsequently activating NF-κB-mediated IFN-I response. Furthermore, the therapeutic potential of Cedrelone lies in its ability to enhance antiviral immunity in primary macrophages and to promote survival and reduce lung tissue damage in HSV-1-infected mice in a PBLD-dependent manner. Consequently, our findings provide a potential combination model that targets PBLD for Cedrelone antiviral drug therapy, potentially paving the way for the development of broad-spectrum antiviral agents.

Interferon regulatory factor 8 (IRF8), an essential member of the IRFs protein family, serves as a critical transcriptional regulator in cytokine signaling, gene transcription, and the differentiation and proliferation of immune cells. However, its function on the bovine ephemeral fever virus (BEFV) infection has not been described. In this study, we demonstrate that BEFV infection upregulates the expression of IRF8, and IRF8 promotes the replication of BEFV. Subsequent investigations reveal that IRF8 suppresses the type I IFN signaling pathway via the degradation of IRF9 in the context of BEFV infection. Mechanistically, IRF8 up-regulates NEDD4 Like E3 ubiquitin ligase (NEDD4L) expression, thereby promoting the IRF9 degradation through the ubiquitin-proteasome pathway. Notably, the inhibitory effect of IRF8 on the BEFV-mediated type I IFN signaling pathway was markedly reduced, and the promoting effect of IRF8 on BEFV replication was attenuated in NEDD4L-knockdown cells, unveiling a novel mechanism by which IRF8-NEDD4L-IRF9 axis hijacks type I interferon signaling pathway to facilitate BEFV infection. These findings board valuable insights into the function of IRF8, which may serve as a basis for the design of novel antiviral agents.

Endoplasmic reticulum (ER) autophagy (ER-phagy) is a vital homeostatic process triggered by multiple signals and plays a crucial role in regulating innate immunity and viral replication. However, the mechanisms by which host proteins utilize ER-phagy to regulate innate immune response during viral infection remains largely unclear. Here, we uncover the regulatory crosstalk between innate immune adapter, ER retention protein Stimulator of Interferon Genes (STING), and the G protein-coupled receptor ADGRE5/CD97 (Cluster of Differentiation 97). Our results demonstrate that CD97 suppresses the STING-mediated type-I interferon (IFN-I) response against DNA virus and cytosolic DNA, thereby promoting herpes simplex virus type 1 (HSV-1) replication in both cells and mice. CD97 facilitates the recruitment of the ER-phagy receptor, FAM134B (family with sequence similarity 134, member B), to initiate ER-phagy, resulting in the degradation of STING subsequent to DNA virus infection. Furthermore, Cd97-deficient mice exhibit higher IFN-I response and greater resistance to HSV-1 infection. Additionally, our findings reveal that inhibiting CD97 with sanguinarine effectively disrupts HSV-1 replication. These findings shed light on the role of CD97 in the innate immune response against DNA virus infections and offer valuable checkpoint for anti-viral STING activation.