Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogenic agents that causes enormous economic losses to the swine industry worldwide (Neumann et al., 2005). It is a member of the family Arteriviridae within the order Nidovirales, and it has an approximately 15.1-kb, single-stranded, positive-sense RNA genome (Gorbalenya et al., 2006). PRRSV is divided into two genotypes: the European genotype (genotype 1) and the North American genotype (genotype 2), which share only approximately 60% identity (Ke and Yoo, 2017). PRRSV causes a series of clinical symptoms, including abortion, premature birth, stillbirth, and mummified fetuses in pregnant sows, and respiratory disturbances in pigs of all ages, especially piglets (Nelsen et al., 1999). However, an epidemic of highly pathogenic PRRSV with high morbidity and high mortality broke out in China in 2006, and caused huge numbers of deaths in swine herds and was not controlled by commercial vaccines (Tian et al., 2007).
The host innate immune system is the first line of defense against virus infection. Interferons (IFNs) are cytokines with broad-spectrum antiviral activity and immune-enhancing functions. Their antiviral activity depends mainly on hundreds of interferon-stimulated genes (ISGs) that encode factors such as bone marrow stromal cell antigen 2, ribonuclease L, dsRNA-dependent protein kinase, myxovirus resistance 2 (Mx2), viperin, and interferon-induced transmembrane protein 1 (Schulz et al., 2010, Chen et al., 2014, Wang et al., 2017). Among these, bone marrow stromal cell antigen2 prevents virus release and traps progeny virions in the plasma membrane of host cells (Fitzpatrick et al., 2010). Ribonuclease L degrades viral RNA to inhibit viral replication (Li et al., 1998). Cholesterol-25-hydroxylase (CH25H) is a reticulum- associated membrane protein that catalyzes the oxidation of cholesterol to 25-hydroxy cholesterol (25HC) (Li et al., 2017). Recent reports revealed that CH25H is an ISG that inhibits various viruses, including enveloped virus, human immunodeficiency virus (Liu et al., 2013), hepatitis C virus (HCV) (Xiang et al., 2015), herpes simplex virus (You et al., 2017), Zika virus (Li et al., 2017), and pseudorabies virus (PRV) (Wang et al., 2017), as well as some non-enveloped viruses, including human rotavirus, poliovirus, and human papillomavirus-16 (Fessler, 2016). It has been reported that the antiviral activity of CH25H is mediated by 25HC, which inhibits membrane fusion, thereby preventing virus entry (Li et al., 2017, Liu et al., 2013). Meanwhile, the expression of CH25H is also upregulated substantially by IFNs and various Toll-like receptor (TLR) ligands in macrophages and dendritic cells (Park and Scott, 2010, Bauman et al., 2009). In addition, metabonomics studies demonstrated that some viral infections can induce upregulation of 25HC (Clark et al., 2012, Blanc et al., 2013), and treatment with 25HC induces expression of the retinoic acid-inducible gene I in macrophages (Li et al., 2017). 25HC is a very important factor in the regulation of lipid metabolism. Insulin-induced gene 2 protein (Insig2), sterol regulatory element-binding proteins (SREBPs), and the SREBP cleavage-activating protein (SCAP) are endoplasmic reticulum membrane proteins. SREBPs are transported from the endoplasmic reticulum to the Golgi membrane by SCAP, and they form active N-SREBPs in the nucleus to regulate sterol biosynthesis (Liu et al., 2015). 25HC independently binds Insig2 to induce these proteins to form a SREBP/Insig2/SCAP complex, and it keeps the complex in the endoplasmic reticulum, resulting in dysregulation of sterol metabolism in cells (Singaravelu et al., 2015). Many viruses require lipid rafts, and lipid biosynthesis is especially important for viral replication, maturation, and secretion. CH25H specifically disrupts HCV NS5A dimer formation to prevent infection (Chen et al., 2014). However, the roles of CH25H and 25HC in PRRSV infection remain unknown.
In the present study, we found that CH25H and its enzyme product 25HC inhibited PRRSV infection by blocking virus entry into Marc-145 monkey kidney cells. A catalytic mutant of CH25H still inhibited PRRSV infection, and 25HC treatment dramatically decreased PRRSV replication in primary porcine alveolar macrophages (PAMs) in vitro. These discoveries provide new evidence that CH25H and 25HC can be used to prevent and control PRRSV infections, which should be helpful for the development of novel antiviral therapies against this virus in the future.
Section snippets
Cells and virus
Marc-145 cells, an epithelial-derived, African green monkey (Chlorocebus sabaeus) kidney cell line, were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (250 U/mL), and streptomycin (250 μg/mL). PAMs were obtained from piglets free of PRRSV as described previously (Zhang et al., 2009) and were cultured in Roswell Park Memorial Institute 1640 (Invitrogen) medium supplemented with 10%
mRNA levels of the gene encoding CH25H are upregulated by IFN-α in Marc-145 cells
To validate whether CH25H could be observed in Marc-145 cells, cells were stimulated with 2 × 104 IU/ml of IFN-α for 4, 6, and 8 h, and CH25H and Mx2 mRNA levels were measured via qRT-PCR. The results showed that CH25H mRNA levels began to increase when the cells were treated with 20,000 IU/ml IFN-α for 8 h (Fig. 1A). In another experiment, cells were stimulated with IFN-α at concentrations of 10,000–50,000 IU/ml individually for 8 h, and CH25H mRNA levels were analyzed via qRT-PCR. The results showed
Discussion
The innate immune response is the robust first line of defense against invading pathogens. Pattern recognition receptors play important roles in recognizing pathogen-associated molecular patterns (Rasmussen et al., 2009, Fang et al., 2016). They recognize foreign nucleic acids and activate type I IFN pathways to induce IFN production, and then IFNs induce many ISGs via the Janus kinase–signal transducer and activator of transcription pathway to interfere with multiple steps in the viral life
Acknowledgments
This work was supported by the National Natural Science Foundation (grant numbers 31672565 and 31230071) for PRRSV immunology, a grant from the Ministry of Agriculture (grant number CARS-36) for Swine Disease Control, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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