Porcine TRIM21 Enhances Porcine Circovirus 2 Infection and Host Immune Responses, But Inhibits Apoptosis of PCV2-Infected Cells

Tripartite motif protein 21 (TRIM21) is an interferon-inducible E3 ligase, containing one RING finger domain, one B-box motif, one coiled-coil domain at the N-terminal, as well as one PRY domain and one SPRY domain at the C-terminal. TRIM21 is expressed in many tissues and plays an important role in systemic autoimmunity. However, TRIM21 plays different roles in different virus infections. In this study, we evaluate the relationship between porcine TRIM21 and PCV2 infection as well as host immune responses. We found that PCV2 infection modulated the expression of porcine TRIM21. TRIM21 can enhance interferons and proinflammatory factors and decrease cellular apoptosis in PCV2-infected cells. These results indicate that porcine TRIM21 plays a critical role in enhancing PCV2 infection, which is a promising target for controlling and developing the treatment of PCV2 infection.

Low Molecular Weight Sulfated Chitosan Efficiently Reduces Infection Capacity of Porcine Circovirus Type 2 (PCV2) In PK15 Cells

Background Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality could act as a efficient antiviral polymer by mimicking PCV2-cell receptor interactions. Methods Sulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM-EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 copy number upon application different molecular weights, sulfate functionalization, and concentration of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to clarify whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection. Results Chi-S significantly reduced genomic copies of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of Chi-S revealed that exerted antiviral activity through impeding viral attachment and penetration into cells. Conclusions These findings help better understanding PCV2-porcine cells interaction and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidate for uses in antiviral therapies against PCV2-associated diseases.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-β induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle’s Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed show a weakened inhibitory effect on IFN-β induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus–associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Porcine circovirus 2 manipulates PERK-ERO1α axis of endoplasmic reticulum in favor of its replication by derepressing viral DNA from HMGB1 sequestration within nuclei

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of ROS and nucleocytoplasmic migration of HMGB1 via PERK activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei, hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical PCVAD mostly results from activation of latent PCV2 infection by confounding factors such as co-infection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals where PCV2 in infected cells might utilize the elevated ROS to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross-talk among ER stress, autophagy/mitophagy and mitochondria-sourced ROS in favor of PCV2 replication.

PCV2 Triggers PK-15 Cell Apoptosis Through the PLC–IP3R–Ca2+ Signaling Pathway

The endoplasmic reticulum (ER) plays an essential role in Ca2+ concentration balance and protein biosynthesis. During infection, the virus needs to complete its life process with the help of ER. At the same time, ER also produces ER stress (ERS), which induces apoptosis to resist virus infection. Our study explored the Ca2+ concentration, ERS, and the apoptosis mechanism after porcine circovirus 2 (PCV2) infection. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and PK-15 cell ER swelling. The colocalization of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in the cytoplasm was observed by laser confocal microscopy. Western blot and quantitative polymerase chain reaction experiments confirmed that PLC and IP3R expression levels increased after PCV2 infection, and Ca2+ concentration in the cytoplasm increased after virus infection. These results suggest that PCV2 infection triggers ERS of PK-15 cells via the PLC–IP3R–Ca2+ signaling pathway to promote the release of intracellular Ca2+ and led to cell apoptosis.

PCV2 trigger apoptosis of PK-15 cells through the PLC-IP3R-Ca2+ signaling pathway

Phospholipase C (PLC) is a key enzyme in the cell membrane. PLC hydrolyses phosphatidylinositol 4, 5-bisphosphate (PIP2) to generateinositol 1,4, 5-triphosphate (IP3) and diacylglycerol (DAG) that regulates a variety of cellular processes. Evidence indicates the pivotal role of PLC and inositol 1,4,5-trisphosphate receptor(IP3R) in influencing Ca2+ release from the endoplasmic reticulum(ER).At the same time, the imbalance of Ca2+ will stimulate endoplasmic reticulum stress(ERS), leading to cell apoptosis. Viral infection could triggers host defense through apoptosis of the infected cells.However, it is not clear how porcine circovirus type 2 (PCV2) induces apoptosis by affecting Ca2+ homeostasis. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and apoptosis.We also found that the ER swelling of PK-15 cells after viral infection by transmission electron microscopy. Furthemore, the activation of PLC-IP3R-Ca2+ signaling enhanced apoptosis in infected PK-15 cells. Taken together,our findings suggest that PCV2 infection trigger ERS of PK-15 cells via the PLC-IP3R-Ca2+ signaling pathway to promoted the release of intracellular Ca2+, and led to cell apoptosis.