Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection

Highlights

• CH25H and 25HC inhibit PRRSV infection through preventing virus entry.

• CH25H inhibition of PRRSV replication does not entirely dependent on catalytic activity.

• 25HC significantly reduces PRRSV infection in PAMs as well as in Marc-145 cells.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded, positive-sense RNA virus of the Arteriviridae family, has become a global health threat for swine. Cholesterol 25-hydroxylase (CH25H) is an enzyme that catalyzes oxidation of cholesterol to 25-hydroxycholesterol (25HC). The purpose of this study was to explore the antiviral activity of CH25H against PRRSV infection. We found that CH25H was induced by interferon-α and PRRSV in Marc-145 monkey kidney cells. In addition, CH25H and 25HC significantly inhibited PRRSV infection by preventing virus entry. A CH25H mutant that exhibited decreased catalytic activity had an antiviral effect against PRRSV. Treatment with 25HC pre-infection or post-infection significantly inhibited PRRSV infection in primary porcine alveolar macrophages. Our results reveal that CH25H is an interferon-stimulated gene and its production of 25HC can be used as a natural antiviral agent to combat PRRSV 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.