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

Hongchao Gou,Shuai Song,Pinpin Chu,Kunli Zhang,Chunling Li,Rujian Cai,Yan Li,Zhibiao Bian,Zhiyong Jiang,Dongxia Yang

doi:10.3389/fmicb.2021.664353

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.

Highlights

Pseudorabies virus (PRV), known as Aujeszky’s disease virus or Suid herpesvirus type 1 (SuHV1), is an enveloped virus with a 143 kb double-stranded linear DNA encoding more than 70 proteins (Klupp et al, 2004)
To analyze the cell death induced by pseudorabies virus (PRV) infection, PK-15 cells were stained with Propidium Iodide (PI) at 12 and 24 h post-infection
These results indicated that both PRV GD-WH and Rong A (RA) strains induced necrotic cell death in PK-15 cells, independent of viral strain differences

Summary

Introduction

Pseudorabies virus (PRV), known as Aujeszky’s disease virus or Suid herpesvirus type 1 (SuHV1), is an enveloped virus with a 143 kb double-stranded linear DNA encoding more than 70 proteins (Klupp et al, 2004). PRV is an infectious agent of disease in multiple mammals, including pigs, ruminants, carnivores, rodents, and so on (Sun et al, 2016). Humans are reported to be infected by PRV (Li et al, 2020; Liu et al, 2020). Depending on the ability of latent infection and reactivation in trigeminal nerves in pigs, PRV mainly circulates in porcine herds and induces severe economic losses (Rziha et al, 1986). During the infection in pigs, PRV primarily infects the epithelial cells and crosses the basement membrane in order to infect all cell types in underlying tissues in a necrotic fashion (Nauwynck et al, 2007). PRV caused cell death of primary porcine epithelial kidney, superior cervical ganglion, and testicle cells (Geenen et al, 2005, 2007). The mechanism by which PRV induces cell death is still unclear

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