FHC, an NS4B-interacting Protein, Enhances Classical Swine Fever Virus Propagation and Acts Positively in Viral Anti-apoptosis

Gui Qian,Yanming Zhang,Huifang Lv,Kangkang Guo,Wang Dong,Tao Wang,Jihui Lin,Qizhuang Lv,Xiaomeng Li,Jing Zhang

doi:10.1038/s41598-018-26777-8

Gui Qian, Yanming Zhang + Show 8 more

Open Access

https://doi.org/10.1038/s41598-018-26777-8

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Abstract

Classical swine fever virus (CSFV), the etiological agent of classical swine fever, causes enormous economic loss to the pig industry. Ferritin heavy chain (FHC) is a notable anti-apoptotic protein, and existing evidence suggests that CSFV cannot induce apoptosis of host cells, however, the role of FHC in CSFV replication remains unclear. In the present study, we found that recombinant lentivirus-mediated knockdown or overexpression of FHC inhibited or enhanced CSFV replication, respectively, indicating a positive role for FHC in CSFV proliferation. Furthermore, interaction between the CSFV NS4B protein and FHC was confirmed by glutathione S-transferase (GST) pull-down, co-immunoprecipitation (co-IP) and confocal imaging assays. In addition, both CSFV replication and NS4B expression upregulated expression of FHC, which counteracts apoptosis by modulating cellular reactive oxygen species (ROS). These results suggest that FHC, an NS4B-interacting protein, enhances CSFV replication and has a positive role in viral anti-apoptosis by regulating ROS accumulation. This work may provide a new perspective for understanding the mechanism of CSFV pathogenesis.

Highlights

Ferritin heavy chain (FHC) is an anti-apoptotic protein that converts Fe[II] to Fe[III] and thereby decreases the reactive oxygen species (ROS) generated from the Fenton reaction[11,12,13]
We verified FHC as a potential NS4B-interacting protein using the yeast two-hybrid assay
glutathione S-transferase (GST) or GST-FHC proteins purified from bacteria transformed with the pGEX-6P-1 or pGEX-GST-FHC plasmid, respectively, were bound to glutathione agarose, and green fluorescent protein (GFP)-NS4B was added to the mixture

Summary

Introduction

FHC is an anti-apoptotic protein that converts Fe[II] to Fe[III] and thereby decreases the reactive oxygen species (ROS) generated from the Fenton reaction[11,12,13]. FHC has been reported to be upregulated by the nuclear factor kappa B (NF-κB) signaling pathway, antagonizing tumor necrosis factor alpha (TNF-α)-mediated apoptosis by suppressing cellular ROS16,17. FHC participates in viral infection of PK-15 cells, and the ORF4 protein of porcine circovirus type 2 antagonizes apoptosis by stabilizing the level of FHC through physical interaction[18]. ROS-dependent RIG-I-like receptor (RLR) signaling, contributing to persistent viral infection of host cells[19], the anti-apoptotic effect of FHC may be involved in CSFV replication. It is necessary to further investigate whether and how the FHC protein is involved in the CSFV life cycle and to reveal the anti-apoptotic effect of FHC on CSFV infection. CSFV replication and NS4B expression inhibited ROS-mediated apoptosis via upregulation of FHC levels

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