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

Shuo Wang,Jianli Shi,Hongbin He,Xiaoyan Wu,Hong Han,Jun Li,Jiaxin Li,Ying Yang,Yao Tian,Zhe Peng,Chen Li,Zhao Wang,Panpan Sun,Shaojian Xu,Chang Liu

doi:10.3389/fmicb.2021.674907

Abstract

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.

Highlights

Porcine circovirus 2 (PCV2) can cause many diseases in the pig herd, including porcine dermatitis and nephrotic syndrome, A2 congenital tremor (A2CT), porcine proliferative and necrotizing pneumonia, and postweaning multisystemic wasting syndrome, etc. (Magar et al, 2000; Thomson et al, 2001; Dupont et al, 2008)
We found that porcine circovirus 2 (PCV2) infection triggers ER stress (ERS) of PK-15 cells by activating the phospholipase C (PLC)–IP3R–Ca2+ signaling pathway to promote the release of intracellular Ca2+ and induce cell apoptosis
The results of the luciferase marker showed that the levels of cytoplasm-free Ca2+ at 24, 48, and 72 h after PCV2 infection were significantly higher in virus-infected cells, compared with uninfected controls, suggesting that PCV2 infection induced the up-regulation of cytoplasm-free Ca2+ (Figure 1) (24 h: 17.5 vs. 100%, 0.01 < P < 0.05; 48 h: 119.6 vs. 100%, 0.01 < P < 0.05; 72 h: 25.7 vs. 100%, 0.01 < P < 0.05)

Summary

Introduction

Porcine circovirus 2 (PCV2) can cause many diseases in the pig herd, including porcine dermatitis and nephrotic syndrome, A2 congenital tremor (A2CT), porcine proliferative and necrotizing pneumonia, and postweaning multisystemic wasting syndrome, etc. (Magar et al, 2000; Thomson et al, 2001; Dupont et al, 2008). High levels of PCV2 viremia and viral load in tissues, granulomatous inflammation, and immunosuppression were considered the symbols of severe PCV2 infection. The exact pathogenic mechanisms of PCV diseases and PCV-associated diseases (PCVD/PCVAD) are currently unknown. Many studies have reported the coinfection between PCV2 and other swine pathogens, such as Haemophilus parasuis, Mycoplasma pneumoniae, and porcine parvovirus, important cofactors that may enhance PCV2 infection and the severity of PCVD/PCVAD (Darwich et al, 2002, 2004; Nielsen et al, 2003). Previous studies found that the activity of phospholipase C (PLC) could increase the concentration of free Ca2+, activating the apoptotic signaling pathway (Malli et al, 2007).

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