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

2020 ◽  
Author(s):  
Shuo Wang ◽  
Chen Li ◽  
Panpan Sun ◽  
Jianli Shi ◽  
Xiaoyan Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Infection ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Porcine Circovirus Type ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Cellular Processes ◽  
Key Enzyme ◽  
Phosphatidylinositol 4

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.

scholarly journals Porcine Circovirus Type 2 Induces ORF3-Independent Mitochondrial Apoptosis via PERK Activation and Elevation of Cytosolic Calcium

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yikai Zhang ◽  
Renjie Sun ◽  
Shichao Geng ◽  
Ying Shan ◽  
Xiaoliang Li ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Initiation Factor ◽  
Porcine Circovirus ◽  
Mitochondrial Apoptosis ◽  
Infected Cells

ABSTRACT Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316–323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS. IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuo Wang ◽  
Chen Li ◽  
Panpan Sun ◽  
Jianli Shi ◽  
Xiaoyan Wu ◽  
...  
Keyword(s):  
Virus Infection ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Protein Biosynthesis ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Intracellular Ca ◽  
Polymerase Chain ◽  
Trisphosphate Receptor

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.

scholarly journals Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1141
Author(s):  
Xingchen Wu ◽  
Xiaoya Wang ◽  
Tengfei Shi ◽  
Le Luo ◽  
Dan Qiao ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Mapk Pathway ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Rep Proteins ◽  
Il10 Promoter ◽  
Later Phase

Porcine circovirus type 2 (PCV2) is one of the major threats to pig farms worldwide. Although PCV2 has been identified to promote IL-10 production, the detailed regulatory roles of PCV2 Rep for IL-10 production remain unclear. Herein, we first found that PCV2 Rep, rather than PCV1 Rep, enhanced IL-10 expression at the later phase of PCV2 infection in porcine alveolar macrophages (PAMs). Furthermore, we found that PCV2 Rep directly activated the p38-MAPK pathway to promote transcription factors NF-κB p50 and Sp1 binding to the il10 promoter, but PCV1 Rep did not. During PCV2 infection, however, PCV2 Rep promoted the binding activities of NF-κB p50 and Sp1 with the il10 promoter only at the later phase of PCV2 infection, since Rep proteins only expressed at the later phase of the infection. Moreover, silence of the thymine DNA glycosylase (TDG), a Rep-binding protein, significantly reduced the binding activities of NF-κB p50 and Sp1 with il10 promoter, resulting in the reduction of IL-10 production in PCV2-inoculated PAMs at the later phase of infection. Taken together, our results demonstrate that Rep proteins enhance IL-10 production during PCV2 infection of PAMs via activation of p38-MAPK pathways, in which host TDG is a critical mediator.

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

2021 ◽  
Author(s):  
Daniela Jiménez-Arriagada ◽  
Alejandro A. Hidalgo ◽  
Victor Neira ◽  
Andrónico Neira-Carrillo ◽  
Sergio A. Bucarey
Keyword(s):  
Molecular Weight ◽  
Antiviral Activity ◽  
Mode Of Action ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Molecular Weights ◽  
Associated Diseases

Abstract 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.

scholarly journals Strain-Dependent Porcine Circovirus Type 2 (PCV2) Entry and Replication in T-Lymphoblasts

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 813 ◽  
Author(s):  
Wei ◽  
Van Renne ◽  
Nauwynck
Keyword(s):  
Nucleic Acid ◽  
Serine Proteases ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Productive Infection ◽  
Porcine Circovirus ◽  
Progeny Virus ◽  
T Lymphoblasts

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases (PCVAD). PCV2 targets lymphoblasts, and pigs suffering from PCVAD display lymphocyte depletion in lymphoid tissues. PCV2 infection of lymphoblasts has not been studied. Here, the replication cycle of PCV2 (abortion strain 1121 and PMWS strain Stoon1010) in T-lymphoblasts was examined. The expression of Rep and Cap were found for both viral strains, while progeny virus was detected for Stoon1010 but not for 1121. PCV2 attached to 11–26% (1121-Stoon1010) of the T-lymphoblasts while 2.6–12.7% of cells showed virus internalization. Chondroitin sulfate (CS) was present on 25% of T-lymphoblasts, and colocalized with PCV2 on 31–32% of the PCV2+ cells. Enzymatic removal of CS reduced PCV2 infection. PCV2 infection was decreased by chlorpromazine, cytochalasin D and Clostridium difficile toxin B for both viral strains and by amiloride for 1121 but not for Stoon1010. Inhibiting either endosome acidification or serine proteases strongly reduced PCV2 infection. Three-dimensional analysis of Cap structure demonstrated a better Cap-nucleic acid affinity for Stoon1010 than for 1121. Taken together, PCV2 binds to T-lymphoblasts partially via CS, enters via clathrin-mediated endocytosis, and disassembles under functions of a pH-drop and serine proteases. Strain Stoon1010 displayed an enhanced viral binding, a specific receptor-mediated endocytosis, an increased Cap-nucleic acid affinity, and a more productive infection in T-lymphoblasts than 1121 did, indicating an evolution from 1121 to Stoon1010.

scholarly journals Identification of lncRNAs Involved in PCV2 Infection of PK-15 Cells

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 479
Author(s):  
Jin He ◽  
Chaoliang Leng ◽  
Jiazhen Pan ◽  
Aoqi Li ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Severe Disease ◽  
Enrichment Analysis ◽  
Economic Loss ◽  
Porcine Circovirus Type ◽  
Pcv2 Infection ◽  
Differentially Expressed ◽  
Porcine Circovirus ◽  
Swine Industry

Porcine circovirus type 2 (PCV2) can cause severe disease in infected pigs, resulting in massive economic loss for the swine industry. Transcriptomic and proteomic approaches have been widely employed to identify the underlying molecular mechanisms of the PCV2 infection. Numerous differentially expressed mRNAs, miRNAs, and proteins, together with their associated signaling pathways, have been identified during PCV2 infection, paving the way for analysis of their biological functions. Long noncoding RNAs (lncRNAs) are important regulators of multiple biological processes. However, little is known regarding their role in the PCV2 infection. Hence, in our study, RNA-seq was performed by infecting PK-15 cells with PCV2. Analysis of the differentially expressed genes (DEGs) suggested that the cytoskeleton, apoptosis, cell division, and protein phosphorylation were significantly disturbed. Then, using stringent parameters, six lncRNAs were identified. Additionally, potential targets of the lncRNAs were predicted using both cis- and trans-prediction methods. Interestingly, we found that the HOXB (Homeobox B) gene cluster was probably the target of the lncRNA LOC106505099. Enrichment analysis of the target genes showed that numerous developmental processes were altered during PCV2 infection. Therefore, our study revealed that lncRNAs might affect porcine embryonic development through the regulation of the HOXB genes.

scholarly journals Porcine Circovirus Type 2 Infection Decreases the Efficacy of a Modified Live Porcine Reproductive and Respiratory Syndrome Virus Vaccine

2006 ◽  
Vol 13 (8) ◽  
pp. 923-929 ◽  
Author(s):  
T. Opriessnig ◽  
N. E. McKeown ◽  
K. L. Harmon ◽  
X. J. Meng ◽  
P. G. Halbur
Keyword(s):  
Age Groups ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Pcv2 Infection ◽  
Daily Weight Gain ◽  
Porcine Circovirus ◽  
Respiratory Syndrome Virus ◽  
Live Virus ◽  
Lymphoid Depletion

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia is a major problem, and vaccination is used to reduce losses associated with PRRSV. Porcine circovirus type 2 (PCV2) causes lymphoid depletion, and there is concern that this adversely affects the immune response. The objective of this study was to investigate the effect of PCV2 infection on the efficacy of modified live virus (MLV) PRRSV vaccine. Sixty-nine 2-week-old pigs were randomly assigned to one of seven groups of 9 to 10 pigs each. At 6 weeks of age, pigs in groups 4, 5, and 6 were inoculated intranasally with PCV2 ISU-40895. At 8 weeks of age, groups 3, 4, 6, and 7 were vaccinated with a PRRSV MLV vaccine. At 12 weeks of age, groups 2, 3, and 4 were challenged with PRRSV SDSU73. All pigs were necropsied 14 days after PRRSV challenge. PCV2-infected, PRRSV-vaccinated, and PRRSV-challenged pigs had significantly (P < 0.05) more-severe macroscopic lung lesions than did the PRRSV-vaccinated and PRRSV-challenged pigs that were not exposed to PCV2 prior to PRRSV vaccination. Nonvaccinated PRRSV-infected pigs had a significantly (P < 0.001) higher incidence of PRRSV antigen in lungs than did all other groups except the group infected with PCV2 prior to PRRSV vaccination and challenge. The nonvaccinated PRRSV-challenged group and the group challenged with PCV2 prior to PRRSV vaccination and challenge had significantly (P < 0.001) lower average daily weight gain than did the control and the vaccinated groups. This work suggests that PCV2 infection has an adverse effect on the development of protective immunity induced by PRRSV vaccine.

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