Abstract
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.
Highlights
The innate immune response is the first line of defense against invading pathogens, by which numerous pattern recognition receptors (PRRs) recognize pathogens, triggering the promotion of the interferon (IFN)-mediated innate immune response and leading to transcription of numerous IFN-stimulated genes (ISGs) [1,2,3]
Recent reports have revealed that 25HC inhibits the replication of various enveloped viruses, including hepatitis C virus (HCV) [16, 17], porcine reproductive and respiratory syndrome virus (PRRSV) [18], murine cytomegalovirus (MCMV) [19], West Nile virus [20], pseudorabies virus (PRV) [21], herpes simplex virus 1 [22], as well as one non-enveloped virus [23], but it is inactive against another non-enveloped virus [19]
The virus stocks of Singapore grouper iridovirus (SGIV) and purified SGIV were propagated in Grouper spleen (GS) cells, while the red-spotted grouper nervous necrosis virus (RGNNV) stocks were propagated in grouper brain (GB) cells, and the titers of the viruses were determined in GS cells and GB cells, respectively, that were both grown in Leibovitz’s L15 medium containing 10% fetal bovine serum (FBS) [32, 38]
Summary
The innate immune response is the first line of defense against invading pathogens, by which numerous pattern recognition receptors (PRRs) recognize pathogens, triggering the promotion of the interferon (IFN)-mediated innate immune response and leading to transcription of numerous IFN-stimulated genes (ISGs) [1,2,3]. Recent reports have revealed that 25HC inhibits the replication of various enveloped viruses, including hepatitis C virus (HCV) [16, 17], porcine reproductive and respiratory syndrome virus (PRRSV) [18], murine cytomegalovirus (MCMV) [19], West Nile virus [20], pseudorabies virus (PRV) [21], herpes simplex virus 1 [22], as well as one non-enveloped virus (poliovirus) [23], but it is inactive against another non-enveloped virus (adenovirus) [19]. Increased evidence demonstrates that 25HC blocks the entry of some viruses, including vesicular stomatitis virus (VSV) [19], human immunodeficiency virus (HIV) [19] and PRRSV [24], by modifying membrane fusion between viruses and cells
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