N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Porcine Epidemic Diarrhea Virus Inhibits HDAC1 Expression To Facilitate Its Replication via Binding of Its Nucleocapsid Protein to Host Transcription Factor Sp1.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.

Establishment of enzyme-linked immunosorbent assays based on recombinant S1 and its truncated proteins for detection of PEDV IgA antibody.

Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21-279), pFastBac1-S1T2 (aa 280-539) and pFastBac1-S1T3 (aa 540-788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

Novel Pattern Recognition Receptor Protects Shrimp by Preventing Bacterial Colonization and Promoting Phagocytosis.

The recognition of pathogen-associated molecular patterns is accomplished by the recognition modules of pattern recognition receptors (PRRs). Leucine-rich repeats (LRRs) and C-type lectin-like domain (CTLD) represent the two most universal categories of recognition modules. In the current study, we identified a novel soluble and bacteria-inducible PRR comprising LRRs and a CTLD from the hepatopancreas of kuruma shrimp Marsupenaeus japonicus and named it Leulectin. The module arrangement of Leulectin is unique among all organisms. Both modules, together with the whole molecule, protected shrimp against Vibrio infection. By screening the pathogen-associated molecular patterns that shrimp might encounter, Leulectin was found to sense Vibrio flagellin through the LRRs and to recognize LPS through CTLD. The LRR-flagellin interaction was confirmed by pull-down and far-Western assays and was found to rely on the fourth LRR of Leulectin and the N terminus of flagellin. The recognition of LPS was determined by the long loop region of CTLD in a calcium-independent manner. By sensing the flagellin, LRRs could prevent its attachment to shrimp cells, thereby inhibiting Vibrio colonization. With the ability to recognize LPS, CTLD could agglutinate the bacteria and promote hemocytic phagocytosis. Our study clearly showed the division of labor and the synergy between different recognition modules and provided new insights into the concept of pattern recognition and the function of soluble PRRs in the antibacterial response.

Thermodynamic and Economic Analyses of Reformative Design for High Back-Pressure Heating in Coal-Fueled Cogeneration Units.

High back-pressure (HBP) heating technology has been identified as an effective approach to improve the efficiency of combined heat and power (CHP). In this study, the novel concept of a HBP heating system with energy cascade utilization is developed and its probability examined. In the reformative design, the extracted heating steam from the intermediate-pressure turbine (IPT) is first drawn to an additional turbine where its excess pressure can be converted into electricity, then steam with a lower pressure can be employed to heat the supply water. As a consequence, the exergy destruction in the supply water heating process can be reduced and the efficiency of the cogeneration unit raised. A detailed thermodynamic investigation was performed based on a typical coal-fired HBP-CHP unit incorporating the proposed configuration. The results show that the artificial thermal efficiency (ATE) promotion was as much as 2.01 percentage points, with an additional net power output of 8.4 MW compared to the reference unit. This was attributed to a 14.65 percentage-point increment in the exergy efficiency of the supply water heating process caused by the suggested retrofitting. The influences of the unit power output, unit heat output, supply water and return water temperatures and turbine back pressure on the thermal performance of the modified system are discussed as well. In addition, the economic performance of the new design is assessed, indicating that the proposed concept is financially feasible.

Selective precipitation and characterization of lignin-carbohydrate complexes (LCCs) from Eucalyptus.

MAIN CONCLUSION: Six types of lignin-carbohydrate complex (LCC) fractions were isolated from Eucalyptus. The acidic dioxane treatment applied significantly improved the yield of LCCs. The extraction conditions had a limited impact on the LCC structures and linkages. Characterization of the lignin-carbohydrate complex (LCC) structures and linkages promises to offer insight on plant cell wall chemistry. In this case, Eucalyptus LCCs were extracted by aqueous dioxane, and then precipitated sequentially by 70% ethanol, 100% ethanol, and acidic water (pH = 2). The composition and structure of the six LCC fractions obtained by selective precipitation were investigated by sugar analysis, molecular weight determination, and 2D HSQC NMR. It was found that the acidic (0.05-M HCl) dioxane treatment significantly improved the yield of LCCs (66.4% based on Klason lignin), which was higher than the neutral aqueous dioxane extraction, and the extraction condition showed limited impact on the LCC structures and linkages. In the fractionation process, the low-molecular-weight LCCs containing a high content of carbohydrates (60.3-63.2%) were first precipitated by 70% ethanol from the extractable solution. The phenyl glycoside (PhGlc) bonds (13.0-17.0 per 100Ar) and highly acetylated xylans were observed in the fractions recovered by the precipitation with 100% ethanol. On the other hand, such xylan-rich LCCs exhibited the highest frequency of ß-O-4 linkages. The benzyl ether (BE) bonds were only detected in the fractions obtained by acidic water precipitation.

Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp.

Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, ΔMjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.

Myeloid leukemia factor functions in anti-WSSV immune reaction of kuruma shrimp, Marsupenaeus japonicus.

Myeloid leukemia factor (MLF) plays an important role in development, cell cycle, myeloid differentiation, and regulates the RUNX transcription factors. However, the function of MLF in immunity is still unclear. In this study, an MLF was identified and characterized in kuruma shrimp Marsupenaeus japonicus, and named as MjMLF. The full-length cDNA of MjMLF contained 1111 nucleotides, which had an opening reading frame of 816 bp encoding a protein of 272 amino acids with an MLF1-interacting protein domain. MjMLF could be ubiquitously detected in different tissues of shrimp at the transcriptional level. The expression pattern analysis showed that MjMLF could be upregulated in shrimp hemocytes and hepatopancreas after white spot syndrome virus challenge. The RNA interference and protein injection assay showed that MjMLF could inhibit WSSV replication in vivo. Flow cytometry assay showed that MjMLF could induce hemocytes apoptosis which functioned in the shrimp antiviral reaction. All the results suggested that MjMLF played an important role in the antiviral immune reaction of kuruma shrimp. The research indicated that MjMLF might function as a novel regulator to inhibit WSSV replication in shrimp.

Suppressor of cytokine signaling 2 (SOCS2) negatively regulates the expression of antimicrobial peptides by affecting the Stat transcriptional activity in shrimp Marsupenaeus japonicus.

The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6 h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V. anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V. anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V. anguillarum at 6 and 12 h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway.

Collaboration between a soluble C-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp.

White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus-associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection-promoting activity of MjsvCL. The MjsvCL-calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection.

A shrimp C-type lectin inhibits proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides.

Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.

Preparation and performance evaluation of a novel orthodontic adhesive incorporating composite dimethylaminohexadecyl methacrylate-Polycaprolactone fibers.

This study addressed enamel demineralization, a common complication in fixed orthodontic treatment, by evaluating a novel orthodontic adhesive with DMAHDM-PCL composite fibers. These fibers, produced through electrospinning, were incorporated into orthodontic adhesive to create experimental formulations at different concentrations and a control group. The study assessed antimicrobial properties, biosafety, and mechanical characteristics. New orthodontic adhesive exhibited significant bacteriostatic effects, reducing bacterial biofilm activity and concentrations. Incorporating 1% and 3% DMAHDM-PCL did not affect cytocompatibility. Animal tests confirmed no inflammatory irritation. Shear bond strength and adhesive residual index results indicated that antimicrobial fibers didn't impact bonding ability. In conclusion, orthodontic adhesives with 3% DMAHDM-PCL fibers are potential antimicrobial bonding materials, offering a comprehensive solution to enamel demineralization in orthodontic patients.

The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Npro of classical swine fever virus enhances HMGB1 acetylation and its degradation by lysosomes to evade from HMGB1-mediated antiviral immunity.

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

Porcine Epidemic Diarrhea Virus Antagonizes Host IFN-λ-Mediated Responses by Tilting Transcription Factor STAT1 toward Acetylation over Phosphorylation To Block Its Activation.

Porcine epidemic diarrhea virus (PEDV) is the main etiologic agent causing acute swine epidemic diarrhea, leading to severe economic losses to the pig industry. PEDV has evolved to deploy complicated antagonistic strategies to escape from host antiviral innate immunity. Our previous study demonstrated that PEDV downregulates histone deacetylase 1 (HDAC1) expression by binding viral nucleocapsid (N) protein to the transcription factor Sp1, inducing enhanced protein acetylation. We hypothesized that PEDV inhibition of HDAC1 expression would enhance acetylation of the molecules critical in innate immune signaling. Signal transducer and activator of transcription 1 (STAT1) is a crucial transcription factor regulating expression of interferon (IFN)-stimulated genes (ISGs) and anti-PEDV immune responses, as shown by overexpression, chemical inhibition, and gene knockdown in IPEC-J2 cells. We further show that PEDV infection and its N protein overexpression, although they upregulated STAT1 transcription level, could significantly block poly(I·C) and IFN-λ3-induced STAT1 phosphorylation and nuclear localization. Western blotting revealed that PEDV and its N protein promote STAT1 acetylation via downregulation of HDAC1. Enhanced STAT1 acetylation due to HDAC1 inhibition by PEDV or MS-275 (an HDAC1 inhibitor) impaired STAT1 phosphorylation, indicating that STAT1 acetylation negatively regulated its activation. These results, together with our recent report on PEDV N-mediated inhibition of Sp1, clearly indicate that PEDV manipulates the Sp1-HDAC1-STAT1 signaling axis to inhibit transcription of OAS1 and ISG15 in favor of its replication. This novel immune evasion mechanism is realized by suppression of STAT1 activation through preferential modulation of STAT1 acetylation over phosphorylation as a result of HDAC1 expression inhibition. IMPORTANCE PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs. We found that PEDV and its N protein inhibit STAT1 phosphorylation and nuclear localization via inducing STAT1 acetylation as a result of HDAC1 downregulation, which, in turn, dampens the host IFN signaling activation. Our study demonstrates a novel mechanism that PEDV evades host antiviral innate immunity through manipulating the reciprocal relationship of STAT1 acetylation and phosphorylation. This provides new insights into the pathogenetic mechanisms of PEDV and even other coronaviruses.

Development and Characterization of Novel Orthodontic Adhesive Containing PCL-Gelatin-AgNPs Fibers.

Enamel demineralization around brackets is a relatively common complication of fixed orthodontic treatment, which seriously affects the aesthetics of teeth. In this study, a novel orthodontic adhesive containing polycaprolactone−gelatin−silver nanoparticles (PCL−gelatin−AgNPs) composite fibers was prepared to prevent enamel demineralization of orthodontic treatment. First, PCL−gelatin−AgNPs fibers film prepared by electrospinning was made into short fibers and added to traditional orthodontic adhesives (Transbond XT, 3M Unitek) in three different ratios to design a series of composite adhesives containing antibacterial materials. The antimicrobial performance of the control product and the three samples were then evaluated by bacterial live/dead staining, colony-forming unit (CFU) counts, tensile bond strength (TBS), and adhesive residue index (ARI) scores. The composite adhesives' antimicrobial properties increased with the increasing content of PCL−gelatin−AgNPs short fibers. The addition of complex antimicrobial fibers to 3M Transbond XT adhesive can significantly reduce the CFU of bacterial biofilms (p < 0.05). The bacterial survival rate on the surface of the specimen decreased with the increase of PCL−gelatin−AgNPs short fibers (p < 0.05). The TBS and ARI values (n = 10) indicated that adding PCL−gelatin−AgNPs short fibers had no significant adverse effect on adhesion. Therefore, adding PCL−gelatin−AgNPs short fibers makes it possible to fabricate orthodontic adhesives with strong antibacterial properties without compromising the bonding ability, which is essential for preventing enamel demineralization around the brackets.