RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells

Abstract

Pigs infected by pseudorabies virus (PRV) display necrotic pathology in multiple organs. The mechanism by which PRV induces cell death is still unclear. Recently, necroptosis was identified as a programmed process dependent on the receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase-like protein (MLKL). In this study, we demonstrated that PRV induced RIPK3-dependent necroptosis in PK-15 cells. The data showed that PRV infection caused cell death with Propidium Iodide (PI)-positive staining. Transmission electron microscopy analysis indicated plasma membrane disruption in PRV-infected cells. A pan-caspase inhibitor did not prevent PRV-induced necrotic cell death. Western blot analysis indicated that caspase-3 and caspase-8 were not cleaved during PRV infection. Although the transcription of tumor necrosis factor-alpha (TNF-α) was increased by PRV infection, RIPK1 was shown to be not involved in PRV-induced necrotic cell death by use of its specific inhibitor. Further experiments indicated that the phosphorylation of RIPK3 and MLKL was upregulated in PRV-infected cells. Stable shRNA knockdown of RIPK3 or MLKL had a recovery effect on PRV-induced necrotic cell death. Meanwhile, viral titers were enhanced in RIPK3 and MLKL knockdown cells. Hence, we concluded that initiation of necroptosis in host cells plays a limiting role in PRV infection. Considering that necroptosis is an inflammatory form of programmed cell death, our data may be beneficial for understanding the necrotic pathology of pigs infected by PRV.

Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF-α

“…In this study, calcein-AM/PI staining and TUNEL staining were used to examine necroptosis and apoptosis in ARPE19 cells, respectively. PI only penetrates damaged cell membranes and releases red fluorescence when embedded in double-stranded DNA, so it has been used to identify necroptotic cells in recent studies [ 37 , 38 ]. TUNEL is a commonly used method to label apoptotic cells by specifically detecting DNA breaks that occur during apoptosis.…”

Targeting Human Proteins for Antiviral Drug Discovery and Repurposing Efforts: A Focus on Protein Kinases

“…Receptor-interacting protein kinases (PIPKs), i.e., RIP kinases, are a family of serine/threonine kinases that play a wide variety of functional roles in cellular signaling during pathogen infection. Several RIPKs are involved in necroptosis, which is a form of inflammatory programmed cell death that plays a role in fighting viral infections [ 124 ]. Necroptosis leads to cell membrane rupture and leakage of cellular contents [ 125 ], thus initiating inflammation and adaptive immunity against viruses [ 126 ].…”

“…Necroptosis also limits Vaccinia virus replication, and RIPK3 deletion in mice caused an increase in virus replication [ 135 ]. Furthermore, a study involving the use of swine kidney cell line (PK-15) and pseudorabies virus (PRV) [ 124 ] reported the induction of necroptosis by PRV via the RIPK3 pathway. Although PRV has induced necroptosis via the RIPK3 pathway in PK-15 cells, the study has shown that RIPK3 and MLKL knockdown had reduced necroptosis and enhanced PRV replication [ 124 ].…”

“…Furthermore, a study involving the use of swine kidney cell line (PK-15) and pseudorabies virus (PRV) [ 124 ] reported the induction of necroptosis by PRV via the RIPK3 pathway. Although PRV has induced necroptosis via the RIPK3 pathway in PK-15 cells, the study has shown that RIPK3 and MLKL knockdown had reduced necroptosis and enhanced PRV replication [ 124 ]. Hence, drugs targeting necroptotic pathways to interfere with viral replication and spreading are worth investigating.…”

The Probiotic Attributes and Anti-pseudorabies Virus Evaluation of Lactobacillus Isolates

“…As an epidemic swine pathogen, pseudorabies virus (PRV) causes significant economic losses to the global swine industry and is characterized by acute and high fever and neurological symptoms in piglets, abortions and stillbirths in sows, and digestive system diseases, such as vomiting, diarrhea, necrotizing enteritis, in adult pigs ( 7 ). Pseudorabies (PR) caused by PRV is a serious infectious disease, with mortality of nearly 100% in piglets ( 8 ). The swine is the natural host for PRV, and the virus can also infect most mammals, including cattle, rodents, and dogs ( 9 ).…”