Canadian Society of Microbiologists’ Armand -Frappier Gold ... › bitstream › 1807 › ... · 41 as potent modulators of cellular metabolism (Brown and Goldstein 1974; Kandutsch

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Canadian Society of Microbiologists’ Armand-Frappier Gold

Medal: The emerging role of 25-hydroxycholesterol in innate immunity

Journal: Canadian Journal of Microbiology

Manuscript ID: cjm-2015-0292.R1

Manuscript Type: Review

Date Submitted by the Author: 07-Jun-2015

Complete List of Authors: Singaravelu, Ragunath; National Research Council Canada, Life Sciences

Division; University of Ottawa, Microbiology and Immunology Srinivasan, Prashanth; National Research Council Canada, Life Sciences Division Pezacki, John; National Research Council Canada, Life Sciences Division

Keyword: 25-hydroxycholesterol, cholesterol-25-hydroxylase, immunity, metabolism, virus

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Canadian Society of Microbiologists’ Armand-Frappier Gold Medal: 1

The emerging role of 25-hydroxycholesterol in innate immunity 2

Ragunath Singaravelu, Prashanth Srinivasan, and John Paul Pezacki 3

R. Singaravelu and P. Srinivasan. Department of Biochemistry, Microbiology and Immunology, Faculty 4

of Medicine, University of Ottawa, Ottawa, ON, Canada, K1N 6N5. Life Sciences Division, National 5

Research Council Canada, Ottawa, ON, Canada, K1A 0R6. 6

J.P. Pezacki. Departments of Chemistry, and Biochemistry, Microbiology and Immunology, University of 7

Ottawa, Ottawa, ON, Canada, K1N 6N5. Life Sciences Division, National Research Council Canada, 8

Ottawa, ON, Canada, K1A 0R6. 9

Corresponding authors: Ragunath Singaravelu (E-mail: [email protected]) and John 10

Paul Pezacki (E-mail: [email protected]) 11

12

Abstract: 13

The metabolic interplay between hosts and viruses plays a crucial role in determining the 14

outcome of viral infection. Viruses re-orchestrate the host’s primary metabolic gene networks, 15

including genes associated with mevalonate and isoprenoid synthesis, to acquire the necessary 16

energy and structural components for their viral life cycles. Recent work has demonstrated that 17

the interferon-mediated antiviral response suppresses the sterol pathway through production of a 18

signalling molecule, 25-hydroxycholesterol (25HC). This oxysterol has been shown to exert 19

multiple effects, both through incorporation into host cellular membranes as well as through 20

transcriptional control. Herein we summarize our current understanding of the multi-functional 21

roles of 25HC in the mammalian innate antiviral response. 22

Keywords: 23

25-hydroxycholesterol, cholesterol-25-hydroxylase, metabolism, virus, immunity 24

25

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Introduction: 26

The importance of different lipid components in physiological processes has been studied for 27

over fifty years. In addition to acting as important structural components (Holthuis and Menon 28

2014), energy stores (Walther and Farese 2012), and post-translational modifications of proteins 29

(Resh 2006), it is becoming increasingly apparent that lipids play important roles as signalling 30

molecules in many biological processes. Since the initial discovery of the eicosanoid 31

prostaglandin as a stimulator of vasodilation (von Euler 1936), numerous additional families of 32

lipid metabolites have been identified as cellular signals, including phosphoinositides, 33

sphingolipids, and fatty acids. Aberrant regulation of these lipid signalling pathways has been 34

linked to several diseased states, such as cancer, chronic inflammation, and metabolic syndrome 35

(reviewed in (Wymann and Schneiter 2008)). Therefore, regulation of the biogenesis, 36

modification, and catabolism of these lipid signalling molecules plays a critical role in the 37

maintenance of physiological processes. 38

Recent work has highlighted oxidized forms of cholesterol, oxysterols, as important signalling 39

molecules. One class of oxysterols, 25-hydroxycholesterols (25HCs), has been long-established 40

as potent modulators of cellular metabolism (Brown and Goldstein 1974; Kandutsch and Chen 41

1975). However, until recently, a clear physiological role has yet to be ascribed to 25HC. 42

Recent work has highlighted a dynamic interplay between host metabolism and innate immunity 43

(Schoggins and Randall 2013). Viruses and other pathogenic organisms (Stehr et al. 2012) 44

perturb the metabolic flux of their host to mediate their proliferation. For example, Su, Pezacki 45

et al. demonstrated that the early host response to hepatitis C virus (HCV) infection involves 46

significant modulated expression of host genes associated with lipid metabolism (Su et al. 2002). 47

Hence, cellular metabolism represents a strategic target pathway for modulation during an 48

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antiviral response. Ghazal and coworkers observed a striking down-regulation in sterol 49

biosynthesis pathway-associated gene expression during the innate antiviral response (Blanc et 50

al. 2013). Subsequent work has elucidated 25HC as the key intermediate signalling molecule 51

which is produced in response to viral infection to mediate this antiviral metabolic effect (Blanc 52

et al. 2013; Liu et al. 2013). This review will summarize our knowledge of the oxysterol’s multi-53

faceted influence on cellular metabolism as it pertains to intrinsic antiviral immunity. 54

25HC: oxysterol regulator of sterol homeostasis 55

Cholesterol and its derivatives represent one of the most abundant lipid species in the human 56

body. Sterol metabolites form a crucial component of cellular membranes and lipid droplets; in 57

addition, farnesylpyrophosphate and geranylpyrophosphate, intermediates from the mevalonate 58

pathway, act as substrates for protein prenylation, an important post-translational modification 59

(Nguyen et al. 2010). Therefore, maintenance of sterol homeostasis represents a critical cellular 60

process. The sterol regulatory element-binding proteins (SREBPs) family of transcription factors 61

are master regulators of lipid biosynthesis. Specifically, SREBP2 promotes expression of genes 62

associated with the mevalonate pathway. There exists a well-established negative feedback 63

mechanism regulating cholesterol biosynthesis, including expression of HMG-CoA reductase 64

(HMGCR), the rate-limiting enzyme (Brown and Goldstein 1980). Newly translated SREBPs 65

bind the SREBP cleavage activating protein (SCAP) at the endoplasmic reticulum (ER). In 66

sterol-depleted conditions, this SREBP/SCAP complex is transported to the Golgi, where 67

SREBP is sequentially cleaved by site-1 protease (S1P) and site-2 protease (S2P) into its mature 68

transcription factor form (Figs. 1a-c). In the presence of excess lipids, cholesterol induces a 69

conformational change in SCAP, which causes binding to INSIGs (Adams et al. 2004), thereby 70

anchoring the SREBP/SCAP complex in the ER and preventing maturation of the transcription 71

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factor. This negative feedback mechanism was one of the first characterized regulatory 72

mechanisms of the sterol pathway (Brown and Goldstein 1999). 73

Prior to 25HC’s discovery as an antiviral lipid effector, it was a well-established suppressor of 74

the sterol pathway. Over 40 years ago, it was discovered that oxidized forms of cholesterol, 75

such as 25HC and 7-ketocholesterol , were able to suppress cholesterol biosynthesis over 100 76

times more potently than unoxidized cholesterol (Brown and Goldstein 1974; Kandutsch and 77

Chen 1975). Subsequent work delineated that cholesterol and oxysterols have independent 78

modes of inhibition of sterol synthesis (Adams et al. 2004) (Fig. 1d). Similar to cholesterol, 79

25HC promotes ER retention of the SREBP/SCAP complex. However, in vitro analysis revealed 80

that 25HC did not directly interact with SCAP (Adams et al. 2004). Given that cells 81

overexpressing INSIG were more sensitive to oxysterol-mediated inhibition of the sterol 82

biosynthesis pathway, this suggested that INSIGs were involved in 25HC’s mode of action. This 83

was subsequently confirmed by work from Goldstein, Brown and colleagues, which revealed that 84

INSIG2 directly binds 25HC and not cholesterol (Radhakrishnan et al. 2007). Binding of 25HC 85

was shown to induce a conformational change in INSIG2, which promotes binding to SCAP and 86

ER retention of SREBPs. Thus, 25HC has been established as a potent repressor of SREBP 87

activation. 88

Independent of its effects on SREBPs, high cellular 25HC levels induce rapid proteasomal 89

degradation of HMGCR (DeBose-Boyd 2008; Lu et al. 2015) (Figs. 1f). Russell-Boyd and 90

coworkers demonstrated that 25HC triggers binding of HMGCR to INSIGs, which results in the 91

accelerated degradation of HMGCR through the ubiquitin-proteasome system (Song et al. 2005). 92

TRC8, MARCH6, and gp78 were identified as three key membrane-anchored E3 ubiquitin-93

protein ligases involved in 25HC stimulated HMGCR degradation (Jo et al. 2011; Song et al. 94

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2005; Zelcer et al.). Subsequently, HMGCR is extracted from the ER membrane into the cytosol 95

in a VCP/p97-dependent manner, enabling HMGCR degradation by the 26S proteasome 96

(Elsabrouty et al. 2013; Morris et al. 2014). Through this mechanism, 25HC inhibits the rate-97

limiting step of cholesterol biosynthesis. 98

The liver X receptors (LXRs) represent another class of transcription factors with pivotal roles in 99

the regulation of cellular lipid homeostasis (Fig. 1e). In vitro studies suggest oxysterols, 100

including 25HC, represent potent endogenous agonists for the LXR pathway (Janowski et al. 101

1996; Lehmann et al. 1997). In the presence of excess sterols, the LXR pathway is a feed-102

forward pathway that promotes cholesterol efflux and bile synthesis for sterol excretion 103

(Lehmann et al. 1997; Peet et al. 1998). 25HC has also been shown to promote cholesterol 104

esterification (Brown et al. 1975; Du et al. 2004), which may result in trafficking of cholesterol 105

from membranes to lipid droplets. Overall, 25HC’s multiple modes of action, highlighted here, 106

should cooperate to decrease cellular sterol content. 107

Cholesterol-25-hydroxylase: an interferon stimulated gene 108

25HC is synthesized by cholesterol-25-hydroxylase (CH25H), an ER-localized multi-109

transmembrane protein expressed in vertebrates (Lund et al. 1998). The enzyme hydroxylates 110

cholesterol in an NADPH-dependent manner. Given the modulatory activity of 25HC in 111

multiple lipid pathways, one would expect CH25H knockout mice to exhibit severe metabolic 112

defects in tolerating high fat diets. While triple-knockout mice for the enzymes synthesizing 113

24S-HC, 27HC, and 25HC demonstrated disrupted LXR signalling (Chen et al. 2007), CH25H 114

deficient mice displayed no aberrant metabolic phenotypes when fed high-fat diets. Collectively, 115

this suggested that these different endogenous oxysterol species have redundant functions in 116

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metabolism. Taken together with the observed low CH25H expression levels in lipogenic tissues 117

(Lund et al. 1998), it also pointed to the likelihood that CH25H (and 25HC) possessed functions 118

independent of its metabolic effects. 119

The first studies highlighting a potential role for CH25H in innate immunity demonstrated that 120

ligand activation of the Toll-like receptor (TLR) family of pathogen recognition receptors 121

induced CH25H expression and 25HC secretion in mouse macrophages and dendritic cells (DCs) 122

(Bauman et al. 2009; Diczfalusy et al. 2009; Park and Scott 2010). Similar increases in CH25H 123

expression were observed in influenza virus infected human airway epithelial cells (Gold et al. 124

2014). TLR activation results in the expression of a family of antiviral cytokines, interferons 125

(IFN), which promotes antiviral gene programming through the Jak/STAT pathway. CH25H 126

expression was demonstrated to be induced by IFN-α and IFN-γ in a Jak1- and STAT1- 127

dependent manner (Blanc et al. 2013; Liu et al. 2013; Park and Scott 2010). Subsequent work 128

demonstrated that interferon regulatory factor 1 (IRF1) cooperates with STAT1 to positively 129

regulate CH25H transcription in mouse primary macrophages (Mboko et al. 2014). Conversely, 130

activating transcription factor 3 (ATF3) (Gold et al. 2012), a known inhibitor of TLR-induced 131

cytokine production, represses CH25H expression. Collectively, these studies pointed towards a 132

role for CH25H-catalyzed 25HC production in the intrinsic pathogen response. 133

25HC: broad antiviral lipid effector 134

There is a broad viral requirement for specific lipid microenvironments to facilitate various 135

stages of viral life cycles (Chukkapalli et al. 2012). Several viruses require altered membrane 136

lipid composition for the formation of replication complexes and lipid envelopes, as well as 137

efficient viral entry and egress. This broad dependence on lipid metabolism is further 138

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exemplified by the fact that small molecule inhibitors of both cholesterol and fatty acid 139

biosynthesis have been demonstrated to elicit antiviral effects against several classes of virus 140

(Blanc et al. 2011; Lyn et al. 2014; Mazièrel et al. 1994; Moser et al. 2012; Munger et al. 2008; 141

Pezacki et al. 2009; Sagan et al. 2006; Su et al. 2002). Prior to the discovery of its role in innate 142

immunity, 25HC was similarly demonstrated to possess broad antiviral activity. The first 143

evidence of 25HC’s antiviral activity derived from the observation that 25-HC and cholesterol 144

treated cells had decreased permissiveness to rhinovirus entry due to down-regulated low density 145

lipoprotein receptor (LDLR) expression (Hofer et al. 1994). Subsequent in vitro studies 146

displayed 25HC’s antiviral effect against viral replication in human immunodeficiency virus 147

(HIV) (Moog et al. 1998), HCV (Pezacki et al. 2009; Su et al. 2002), and West Nile virus 148

(WNV) (Mackenzie et al. 2007) models. Collectively, this work established 25HC as a broad 149

antiviral oxysterol. 150

Two recent companion studies elegantly established CH25H-mediated 25HC production as a 151

critical arm of the mammalian innate antiviral response. Blanc et al. performed supernatant 152

oxysterol profiling of interferon-stimulated and virus-infected mouse macrophages – revealing 153

25HC as the only secreted and intracellular oxysterol that was up-regulated (Blanc et al. 2013). 154

In parallel, Liu et al. also confirmed 25HC as a soluble antiviral factor produced during IFN 155

stimulation of macrophages (Liu et al. 2013). Both studies confirmed broad antiviral activity of 156

25HC in vitro against different classes of lipid enveloped RNA and DNA viruses. This was 157

subsequently reaffirmed by several independent studies (Arita et al. 2013; Chen et al. 2014; 158

Mboko et al. 2014), and the antiviral activity was additionally extended to non-enveloped viruses 159

(Civra et al. 2014). Table 1 summarizes the viruses whose susceptibility to 25HC’s antiviral 160

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effects has been validated. Macrophage and dendritic cell secretion of 25HC should enable both 161

paracrine and autocrine effects, suggesting a more systemic antiviral role for 25HC. 162

Limited in vivo studies have been performed examining 25HC’s antiviral role. To date, only two 163

studies have examined the oxysterol’s relevance to innate immunity in vivo. CH25H -/-

mice 164

were demonstrated to possess increased susceptibility to MHV68 infection (Liu et al. 2013). The 165

same study demonstrated that 25HC treatment of humanized mice prior to infection inhibits HIV 166

replication (Liu et al. 2013). In a separate conflicting report, deletion of CH25H resulted in no 167

significant change in influenza A viral loads (Gold et al. 2014). These contradictory results 168

suggest a potential virus-specific antiviral effect of 25HC in vivo. They also highlight the need 169

for further small animal studies to delineate 25HC’s physiological relevance in the innate 170

antiviral response. 171

25HC: multiple potential modes of action 172

25HC appears to exert effects in a multitude of signalling pathways, including the 173

aforementioned SREBP and LXR signalling pathways. While SREBP signalling appeared to 174

contribute to 25HC’s viral inhibition, these antiviral effects appear to occur in an LXR-175

independent manner (Blanc et al. 2013). Below, we discuss the contribution of repressed 176

SREBP signalling and other pathways to 25HC’s effect on the host response to viral infection. 177

Figure 2 summarizes these effects in the context of HCV infection. The relevance of each of 178

these modes of inhibition appears to be context-specific, depending on the virus as well as on the 179

pre-existing cellular lipid environment. 180

Modification of cellular membranes 181

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While previous work described an antiviral effect for 25HC at the stage of viral replication, Liu 182

and coworkers demonstrated that 25HC also broadly inhibits viral entry (Liu et al. 2013). The 183

authors demonstrated that longer pre-treatment of cells with 25HC results in strong inhibition of 184

vesicular stomatitis virus (VSV) infection. This suggested that 25HC induces an antiviral 185

cellular state prior to infection. After showing a lack of effect on virion attachment, 25HC was 186

determined to inhibit VSV virion fusion to the host cell. This was attributed to changes in the 187

cellular membrane properties, as 25HC treatment also inhibited Nipah virus (NiV) F and G 188

protein-mediated cell-cell fusion. These results were also extended to HIV entry, demonstrating 189

that 25HC modulates membrane properties to inhibit both pH-dependent (VSV) and pH-190

independent (HIV and NiV) modes of viral fusion. The efficiency of virion fusion is sensitive to 191

host membrane lipid composition and fluidity. For example, HIV has been shown to require 192

cholesterol-rich microdomains to facilitate viral entry (Carter et al. 2009). 25HC-mediated 193

suppression of SREBP activation and induction of HMGCR degradation could, therefore, 194

contribute to decreased rates of viral entry. 195

Furthermore, 25HC has been shown to modulate membrane fluidity and cholesterol accessibility 196

(Bielska et al. 2014; Richert et al. 1984), which may contribute to the observed decrease in 197

membrane fluidity in interferon treated cells (Chatterjee et al. 1982). Given that small molecule 198

inhibitors of fusion demonstrate broad antiviral activity against enveloped viruses (St.Vincent et 199

al. 2010), 25HC’s effect on the plasma and endosomal membranes represents a potential 200

contributing mechanism to its antiviral effects. Similarly, modulation of intracellular membranes 201

should result in an antiviral effect against positive RNA viruses, which are known to induce 202

significant ER, Golgi, lysosome, or endosome membrane remodelling to create scaffolds that 203

serve as sites of viral replication (Miller and Krijnse-Locker 2008). Direct integration of 25HC 204

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into these intracellular membranes could restrict the ability of viruses to properly house their 205

replication complexes, while indirect 25HC-induced changes in lipid components of both host 206

membranes and virion envelopes could impair the viruses’ ability to infect bystander cells. 207

Interactions with oxysterol binding proteins 208

The oxysterol binding protein (OSBP) and OSBP related proteins (ORPs) represent another 209

family of proteins which can interact with 25HC. This family of proteins bind and transfer 210

sterols between cellular organelles, such as the Golgi and ER membranes (Mesmin et al. 2013). 211

Interestingly, RNA viruses have been shown to co-opt OSBP and ORPs to facilitate assembly 212

(Amako et al. 2009) and/or to shuttle and enrich cholesterol at sites of viral replication (Barajas 213

et al. 2014; Strating et al. 2015; Wang et al. 2014). In fact, small molecule targeting of OSBP 214

has shown broad-range efficacy in inhibition of enterovirus replication (Strating et al. 2015). In 215

the absence of viral infection, 25HC relocalizes OSBP from the cytoplasm to the Golgi 216

(Ridgway et al. 1992). This has been linked to 25HC’s antiviral effect towards rhinoviruses, 217

where 25HC prevents OSBP-mediated shuttling of cholesterol to the membranous scaffolds of 218

viral replication (Roulin et al. 2014). It remains unknown whether OSBP or any of the ORPs are 219

capable of mediating transfer of 25HC directly to the membranes modified by viruses for 220

replication and particle assembly. 221

Repression of protein prenylation 222

Prenylated host and viral proteins have also been implicated in the replication of viruses, 223

including HCV, hepatitis delta virus, pseudorabies virus, and respiratory syncytial virus (Bordier 224

et al. 2003; Gower and Graham 2001; Kapadia and Chisari 2005; Wang et al. 2005) . Since 225

repression of SREBP signalling should result in down-regulation of cellular farnesyl and 226

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geranylgeranyl pyrophosphate levels, 25HC should elicit alterations in protein prenylation status, 227

which may convey an alternative mechanism of viral inhibition. This is consistent with recent 228

work demonstrating that supplementation with geranylgeraniol can rescue the antiviral effects of 229

lower concentrations of 25HC against murine cytomegalovirus (Blanc et al. 2013). 230

Integrated stress response 231

Transcriptome profiling of 25HC-treated macrophages revealed that, independent of its effect on 232

LXR and SREBP signalling, 25HC could activate genes associated with the integrated stress 233

response (ISR) in macrophages (Shibata et al. 2014). Activation of this pathway was attributed 234

to altered amino acid metabolism and increased oxidative stress and results in suppression of 235

translation, which may contribute to 25HC’s broad antiviral activity. 236

Potentiation of interferon signalling 237

A recent study demonstrated that 25HC potentiates IFN-β expression in primary human 238

bronchial epithelial cells stimulated with poly(I:C), a viral RNA analogue (Koarai et al. 2012). 239

This was partially attributed to increased nuclear translocation of a key transcription factor, IFN 240

regulatory factor 3 (IRF3). In addition, the study demonstrated that 25HC increased nuclear 241

factor-kappa B (NF-κB) signalling, another key regulator of type I and III IFN expression 242

(Ichikawa et al. 2013; Onoguchi et al. 2007). It remains to be seen whether this IFN/25HC-243

mediated feed-forward loop is conserved in other cell types and in vivo. 244

Effects on inflammation and adaptive immunity 245

In addition to its effects on innate immunity, recent studies have proposed a role for 25HC in the 246

regulation of inflammation and adaptive immunity. Gold and colleagues identified pro-247

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inflammatory effects of CH25H-catalyzed 25HC production in mouse macrophages (Gold et al. 248

2014). This activation is mediated, in part, by increased recruitment of activator protein 1 (AP-249

1) transcription factor complex to the promoters of TLR-responsive genes. Accordingly, 250

influenza A virus-infected CH25H knockout mice presented with lower morbidity due to 251

decreased inflammation. Another 25HC-mediated pro-inflammatory effect may result from its 252

activation of NF-kB signalling, resulting in increased release of interleukin (IL)-6 and IL-8 253

(Koarai et al. 2012; Rydberg et al. 2003). Type I IFN signalling has previously been linked to 254

decreased inflammasome activity and production of the pro-inflammatory cytokine IL-1β; 255

however, the ISG responsible for this effect on IL-1β signalling was unclear until a recent study 256

observed increased IL-1β expression levels in CH25H knockout mice (Reboldi et al. 2014). The 257

authors identified IL-1β expression as a SREBP target gene in macrophages, and CH25H as the 258

key ISG responsible for IFN-mediated repression of IL-1β inflammasome activation. 25HC-259

mediated activation of LXR signalling may also negatively influence inflammation via LXR-260

mediated trans-repression (Joseph et al. 2003; Ogawa et al. 2005); however, it remains to be seen 261

if this represents a physiologically relevant mechanism of action in vivo for 25HC. Collectively, 262

these studies illustrate a complex role for 25HC in the regulation of inflammation. 263

There have also been a few studies investigating 25HC’s influence on adaptive immunity that 264

have highlighted a repressive role for the oxysterol. Macrophage-secreted 25HC suppressed 265

both immunoglobulin A (IgA) class switching in B cells as well as IL-2-stimulated B cell 266

proliferation (Bauman et al. 2009). This repressive role for 25HC in adaptive immunity is also 267

consistent with SREBP signalling’s critical role in CD8+ T cell clonal expansion during viral 268

infection (Bensinger et al. 2008; Kidani et al. 2013). 269

Future Outlook 270

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It should be noted that 25HC is metabolized by several other host enzymes; therefore, it is 271

important to consider the roles of these other enzymes and modified oxysterols in the antiviral 272

response. For example, CYP7B1 hydroxylates 25HC to form 7α25HC, an oxysterol which also 273

plays an important role as a guidance cue for B cells through interaction with Epstein-Barr virus 274

induced receptor 2 (EBI2) (reviewed in (Daugvilaite et al. 2014)). Recent work has also 275

identified hydroxysterol sulfotransferase 2B1b (SULT2B1b) as the enzyme which catalyzes 276

sulfation of 25HC to 25-hydroxycholesterol-3-sulfate (25HC3S) (Ren and Ning 2014). Studies 277

have generally found the sulfated oxysterol to have effects opposing to 25HC on both 278

inflammation and metabolism (Ren and Ning 2014). While metabolomic profiling revealed no 279

significant increase in sulfated oxysterol levels during interferon stimulation of macrophages 280

(Blanc et al. 2013), it remains to be seen if 25HC3S plays a role in the antiviral response in other 281

cell types and tissues, such as the liver, where 25HC3S has been shown to be localized in the 282

nuclei and mitochondria (Ren et al. 2006). Future work should consider the importance of 25HC 283

as an intermediate metabolite for other oxysterols which may be key players in immunity. 284

In summary, our understanding of 25HC has rapidly evolved from its initial use as a research 285

tool to its discovery as a natural product. To date, a large body of evidence has supported a key 286

role for 25HC in regulating the crosstalk between metabolism and immunity, however, several 287

questions remain. This IFN-regulated oxysterol has been clearly demonstrated to possess 288

antiviral activity against several classes of viruses, however, we are only beginning to understand 289

how 25HC elicits these broad antiviral effects. The physiological contribution of each of the 290

25HC linked pathways (LXR, SREBP, AP1, OSBP and ISR) to the oxysterol’s role in the 291

antiviral response should be analyzed in vivo. The effect of 25HC on transcription has, thus far, 292

been focused on coding genes. It remains to be seen whether the oxysterol elicits any antiviral 293

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effects through modulation of the non-coding transcriptome. Furthermore, the majority of 294

experiments looking at 25HC have been performed in both in vitro and in vivo mouse models. 295

Patients with HCV chronic infection possess elevated serum levels of 25HC and other oxysterols 296

(Ikegami et al. 2014), however, it is unclear if this was a direct effect of the virus, or a product of 297

autoxidation or the metabolic disease associated with HCV infection (Ikegami et al. 2012). 298

Future work should determine the relevance of CH25H and 25HC to human viral immunity. 299

Acknowledgements 300

Due to space restrictions, we apologize to authors whose work was not cited in this review. R.S. 301

would like to thank the Canadian Society of Microbiology (CSM) for the Armand-Frappier 302

Outstanding Graduate student award, along with the NSERC Vanier Scholarship and the Ontario 303

Graduate Scholarship (OGS) for support in the form of funding. J.P.P. would like to thank the 304

Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering 305

Research Council of Canada (NSERC) for funding in the form of operating grants. Lastly, R.S. 306

would like to thank the National Canadian Research Training Program in Hepatitis C (NCRTP-307

HepC) for additional training and support. 308

309

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References 310

Adams, C.M., Reitz, J., De Brabander, J.K., Feramisco, J.D., Li, L., Brown, M.S., and Goldstein, 311

J.L. 2004. Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different 312

mechanisms, both involving SCAP and Insigs. J. Biol. Chem. 279(50): 52772-52780. doi: 313

10.1074/jbc.M410302200. PMID: 15452130. 314

Amako, Y., Sarkeshik, A., Hotta, H., Yates, J., and Siddiqui, A. 2009. Role of oxysterol binding 315

protein in hepatitis C virus infection. J. Virol. 83(18): 9237-9246.doi: 10.1128/jvi.00958-09. 316

PMID: 19570870. 317

Arita, M., Kojima, H., Nagano, T., Okabe, T., Wakita, T., and Shimizu, H. 2013. Oxysterol-318

binding protein family is the trget of minor enviroxime-like compounds. J. Virol. 87(8): 319

4252-4260. doi: 10.1128/jvi.03546-12. PMID: 23365445. 320

Barajas, D., Xu, K., de Castro Martín, I.F., Sasvari, Z., Brandizzi, F., Risco, C., and Nagy, P.D. 321

2014. Co-opted oxysterol-binding ORP and VAP Proteins channel sterols to RNA virus 322

replication sites via membrane contact sites. PLoS Pathog. 10(10): e1004388. PMID: 323

25329172. 324

Bauman, D.R., Bitmansour, A.D., McDonald, J.G., Thompson, B.M., Liang, G., and Russell, 325

D.W. 2009. 25-hydroxycholesterol secreted by macrophages in response to Toll-like 326

receptor activation suppresses immunoglobulin A production. Proc. Natl. Acad. Sci. U.S.A. 327

106(39): 16764-16769. doi: 10.1073/pnas.0909142106. PMID: 19805370. 328

Bensinger, S.J., Bradley, M.N., Joseph, S.B., Zelcer, N., Janssen, E.M., Hausner, M.A., Shih, R., 329

Parks, J.S., Edwards, P.A., Jamieson, B.D., and Tontonoz, P. 2008. LXR signaling couples 330

sterol metabolism to proliferation in the acquired immune response. Cell 134(1): 97-111. 331

doi: 10.1016/j.cell.2008.04.052. PMID: 18614014. 332

Page 15 of 33



Draft

Bielska, A.A., Olsen, B.N., Gale, S.E., Mydock-McGrane, L., Krishnan, K., Baker, N.A., 333

Schlesinger, P.H., Covey, D.F., and Ory, D.S. 2014. Side-chain oxysterols modulate 334

cholesterol accessibility through membrane remodeling. Biochemistry 53(18): 3042-3051. 335

doi: 10.1021/bi5000096. PMID: 24758724. 336

Blanc, M., Hsieh, W.Y., Robertson, K.A., Kropp, K.A., Forster, T., Shui, G., Lacaze, P., 337

Watterson, S., Griffiths, S.J., Spann, N.J., Meljon, A., Talbot, S., Krishnan, K., Covey, D.F., 338

Wenk, M.R., Craigon, M., Ruzsics, Z., Haas, J., Angulo, A., Griffiths, W.J., Glass, C.K., 339

Wang, Y., and Ghazal, P. 2013. The transcription factor STAT-1 couples macrophage 340

synthesis of 25-hydroxycholesterol to the interferon antiviral response. Immunity 38(1): 341

106-118. PMID: 23273843. 342

Blanc, M., Hsieh, W.Y., Robertson, K.A., Watterson, S., Shui, G., Lacaze, P., Khondoker, M., 343

Dickinson, P., Sing, G., Rodríguez- Martín, S., Phelan, P., Forster, T., Strobl, B., Müller, M., 344

Riemersma, R., Osborne, T., Wenk, M.R., Angulo, A., and Ghazal, P. 2011. Host defense 345

against viral infection involves interferon mediated down-regulation of sterol biosynthesis. 346

PLoS Biol. 9(3): e1000598. PMID: 21408089. 347

Bordier, B.B., Ohkanda, J., Liu, P., Lee, S.Y., Salazar, F.H., Marion, P.L., Ohashi, K., Meuse, 348

L., Kay, M.A., Casey, J.L., Sebti, S.M., Hamilton, A.D., and Glenn, J.S. 2003. In vivo 349

antiviral efficacy of prenylation inhibitors against hepatitis delta virus. J. Clin. Invest. 350

112(3): 407-414. doi: 10.1172/jci17704. PMID: 12897208. 351

Brown, M.S., Dana, S.E., and Goldstein, J.L. 1975. Cholesterol ester formation in cultured 352

human fibroblasts. Stimulation by oxygenated sterols. J. Biol. Chem. 250(10): 4025-4027. 353

PMID: 1126942. 354

Page 16 of 33



Draft

Brown, M.S., and Goldstein, J.L. 1974. Suppression of 3-hydroxy-3-methylglutaryl coenzyme A 355

reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J. 356

Biol. Chem. 249(22): 7306-7314. PMID: 4436312. 357

Brown, M.S., and Goldstein, J.L. 1980. Multivalent feedback regulation of HMG CoA reductase, 358

a control mechanism coordinating isoprenoid synthesis and cell growth. J. Lipid Res. 21(5): 359

505-517. PMID: 6995544. 360

Brown, M.S., and Goldstein, J.L. 1999. A proteolytic pathway that controls the cholesterol 361

content of membranes, cells, and blood. Proc. Natl. Acad. Sci. U.S.A. 96(20): 11041-11048. 362

doi: 10.1073/pnas.96.20.11041. PMID: 10500120. 363

Carter, G.C., Bernstone, L., Sangani, D., Bee, J.W., Harder, T., and James, W. 2009. HIV entry 364

in macrophages is dependent on intact lipid rafts. Virology 386(1): 192-202. doi: 365

10.1016/j.virol.2008.12.031. PMID: 19185899. 366

Chatterjee, S., Cheung, H.C., and Hunter, E. 1982. Interferon inhibits Sendai virus-induced cell 367

fusion: an effect on cell membrane fluidity. Proc. Natl. Acad. Sci. U.S.A. 79(3): 835-839. 368

PMID: 6174982. 369

Chen, W., Chen, G., Head, D.L., Mangelsdorf, D.J., and Russell, D.W. 2007. Enzymatic 370

reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice. Cell 371

Metab. 5(1): 73-79. doi: 10.1016/j.cmet.2006.11.012. PMID: 6174982. 372

Chen, Y., Wang, S., Yi, Z., Tian, H., Aliyari, R., Li, Y., Chen, G., Liu, P., Zhong, J., Chen, X., 373

Du, P., Su, L., Qin, F.X., Deng, H., and Cheng, G. 2014. Interferon-inducible cholesterol-374

25-hydroxylase inhibits hepatitis C virus replication via distinct mechanisms. Sci. Rep. 4: 375

7242. doi: 10.1038/srep07242. PMID: 25467815. 376

Page 17 of 33



Draft

Chukkapalli, V., Heaton, N.S., and Randall, G. 2012. Lipids at the interface of virus-host 377

interactions. Curr. Opin. Microbiol. 15(4): 512-518. doi: 10.1016/j.mib.2012.05.013. 378

PMID: 22682978. 379

Civra, A., Cagno, V., Donalisio, M., Biasi, F., Leonarduzzi, G., Poli, G., and Lembo, D. 2014. 380

Inhibition of pathogenic non-enveloped viruses by 25-hydroxycholesterol and 27-381

hydroxycholesterol. Sci. Rep. 4: 7487. doi: 10.1038/srep07487. PMID: 25501851. 382

Daugvilaite, V., Arfelt, K.N., Benned-Jensen, T., Sailer, A.W., and Rosenkilde, M.M. 2014. 383

Oxysterol-EBI2 signaling in immune regulation and viral infection. Eur. J. Immunol. 44(7): 384

1904-1912. doi: 10.1002/eji.201444493. PMID: 24810762. 385

DeBose-Boyd, R.A. 2008. Feedback regulation of cholesterol synthesis: sterol-accelerated 386

ubiquitination and degradation of HMG CoA reductase. Cell Res. 18(6): 609-621. doi: 387

10.1038/cr.2008.61. PMID: 18504457. 388

Diczfalusy, U., Olofsson, K.E., Carlsson, A.-M., Gong, M., Golenbock, D.T., Rooyackers, O., 389

Fläring, U., and Björkbacka, H. 2009. Marked upregulation of cholesterol 25-hydroxylase 390

expression by lipopolysaccharide. J. Lipid Res. 50(11): 2258-2264. doi: 391

10.1194/jlr.M900107-JLR200. PMID: 19502589. 392

Du, X., Pham, Y.H., and Brown, A.J. 2004. Effects of 25-hydroxycholesterol on cholesterol 393

esterification and sterol regulatory element-binding protein processing are dissociable: 394

implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum. 395

J. Biol. Chem. 279(45): 47010-47016. doi: 10.1074/jbc.M408690200. PMID: 15317807. 396

Elsabrouty, R., Jo, Y., Dinh, T.T., and DeBose-Boyd, R.A. 2013. Sterol-induced dislocation of 397

3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells. 398

Mol. Biol. Cell 24(21): 3300-3308. doi: 10.1091/mbc.E13-03-0157. PMID: 24025715. 399

Page 18 of 33



Draft

Gold, E.S., Diercks, A.H., Podolsky, I., Podyminogin, R.L., Askovich, P.S., Treuting, P.M., and 400

Aderem, A. 2014. 25-hydroxycholesterol acts as an amplifier of inflammatory signaling. 401

Proc. Natl. Acad. Sci. U.S.A. 111(29): 10666-10671. doi: 10.1073/pnas.1404271111. PMID: 402

24994901. 403

Gold, E.S., Ramsey, S.A., Sartain, M.J., Selinummi, J., Podolsky, I., Rodriguez, D.J., Moritz, 404

R.L., and Aderem, A. 2012. ATF3 protects against atherosclerosis by suppressing 25-405

hydroxycholesterol-induced lipid body formation. J. Exp. Med. 209(4): 807-817. doi: 406

10.1084/jem.20111202. PMID: 22473958. 407

Gower, T.L., and Graham, B.S. 2001. Antiviral activity of lovastatin against respiratory syncytial 408

virus in vivo and in vitro. Antimicrob. Agents and Chemother. 45(4): 1231-1237. doi: 409

10.1128/aac.45.4.1231-1237.2001. PMID: 11257039. 410

Hofer, F., Gruenberger, M., Kowalski, H., Machat, H., Huettinger, M., Kuechler, E., and Blass, 411

D. 1994. Members of the low density lipoprotein receptor family mediate cell entry of a 412

minor-group common cold virus. Proc. Natl. Acad. Sci. U.S.A. 91(5): 1839-1842. doi: 413

10.1073/pnas.91.5.1839. PMID: 8127891. 414

Holthuis, J.C.M., and Menon, A.K. 2014. Lipid landscapes and pipelines in membrane 415

homeostasis. Nature 510(7503): 48-57. doi: 10.1038/nature13474. PMID: 24899304. 416

Ichikawa, T., Sugiura, H., Koarai, A., Kikuchi, T., Hiramatsu, M., Kawabata, H., Akamatsu, K., 417

Hirano, T., Nakanishi, M., Matsunaga, K., Minakata, Y., and Ichinose, M. 2013. 25-418

hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB 419

dependent pathway. Exp. Cell Res. 319(8): 1176-1186. doi: 10.1016/j.yexcr.2013.02.014. 420

PMID: 23485764. 421

Page 19 of 33



Draft

Ikegami, T., Honda, A., Miyazaki, T., Kohjima, M., Nakamuta, M., and Matsuzaki, Y. 2014. 422

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection. 423

Biochem. Biophys. Res. Comm. 446(3): 736-740. doi: 10.1016/j.bbrc.2014.01.176. PMID: 424

24525121. 425

Ikegami, T., Hyogo, H., Honda, A., Miyazaki, T., Tokushige, K., Hashimoto, E., Inui, K., 426

Matsuzaki, Y., and Tazuma, S. 2012. Increased serum liver X receptor ligand oxysterols in 427

patients with non-alcoholic fatty liver disease. J. Gastroenterol. 47(11): 1257-1266. doi: 428

10.1007/s00535-012-0585-0. PMID: 22569763. 429

Janowski, B.A., Willy, P.J., Devi, T.R., Falck, J.R., and Mangelsdorf, D.J. 1996. An oxysterol 430

signalling pathway mediated by the nuclear receptor LXR alpha. Nature 383(6602): 728-431

731. PMID: 8878485. 432

Jo, Y., Lee, P.C.W., Sguigna, P.V., and DeBose-Boyd, R.A. 2011. Sterol-induced degradation of 433

HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and 434

Trc8. Proc. Natl. Acad. Sci. U.S.A. 108(51): 20503-20508. doi: 10.1073/pnas.1112831108. 435

PMID: 22143767. 436

Joseph, S.B., Castrillo, A., Laffitte, B.A., Mangelsdorf, D.J., and Tontonoz, P. 2003. Reciprocal 437

regulation of inflammation and lipid metabolism by liver X receptors. Nat. Med. 9(2): 213-438

219. PMID: 12524534. 439

Kandutsch, A.A., and Chen, H.W. 1975. Regulation of sterol synthesis in cultured cells by 440

oxygenated derivatives of cholesterol. J. Cell. Physiol. 85(S1): 415-424. doi: 441

10.1002/jcp.1040850408. PMID: 164478. 442

Page 20 of 33



Draft

Kapadia, S.B., and Chisari, F.V. 2005. Hepatitis C virus RNA replication is regulated by host 443

geranylgeranylation and fatty acids. Proc. Natl. Acad. Sci. U.S.A. 102(7): 2561-2566. doi: 444

10.1073/pnas.0409834102. PMID: 15699349. 445

Kidani, Y., Elsaesser, H., Hock, M.B., Vergnes, L., Williams, K.J., Argus, J.P., Marbois, B.N., 446

Komisopoulou, E., Wilson, E.B., Osborne, T.F., Graeber, T.G., Reue, K., Brooks, D.G., and 447

Bensinger, S.J. 2013. Sterol regulatory element-binding proteins are essential for the 448

metabolic programming of effector T cells and adaptive immunity. Nat. Immunol. 14(5): 449

489-499. doi: 10.1038/ni.2570. PMID: 23563690. 450

Koarai, A., Yanagisawa, S., Sugiura, H., Ichikawa, T., Kikuchi, T., Furukawa, K., Akamatsu, K., 451

Hirano, T., Nakanishi, M., Matsunaga, K., Minakata, Y., and Ichinose, M. 2012. 25-452

hydroxycholesterol enhances cytokine release and toll-like receptor 3 response in airway 453

epithelial cells. Respir. Res. 13(1): 63. PMID: 22849850. 454

Lehmann, J.M., Kliewer, S.A., Moore, L.B., Smith-Oliver, T.A., Oliver, B.B., Su, J. L., 455

Sundseth, S.S., Winegar, D.A., Blanchard, D.E., Spencer, T.A., and Willson, T.M. 1997. 456

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response 457

pathway. J. Biol. Chem. 272(6): 3137-3140. doi: 10.1074/jbc.272.6.3137. PMID: 9013544. 458

Liu, S.Y., Aliyari, R., Chikere, K., Li, G., Marsden, M.D., Smith, J.K., Pernet, O., Guo, H., 459

Nusbaum, R., Zack, J.A., Freiberg, A.N., Su, L., Lee, B., and Cheng, G. 2013. Interferon-460

inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-461

hydroxycholesterol. Immunity 38(1): 92-105. doi: 10.1016/j.immuni.2012.11.005. PMID: 462

23273844. 463

Lu, H., Talbot, S., Robertson, K.A., Watterson, S., Forster, T., Roy, D., and Ghazal, P. 2015. 464

Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon- 465

Page 21 of 33



Draft

in primary macrophages requires endogenous 25-hydroxycholesterol synthesis. Steroids. In 466

press. doi: 10.1016/j.steroids.2015.02.022. PMID: 25759117. 467

Lund, E.G., Kerr, T.A., Sakai, J., Li, W.P., and Russell, D.W. 1998. cDNA cloning of mouse and 468

human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent 469

oxysterol regulator of lipid metabolism. J. Biol. Chem. 273(51): 34316-34327. doi: 470

10.1074/jbc.273.51.34316. PMID: 9852097. 471

Lyn, R.K., Singaravelu, R., Kargman, S., O'Hara, S., Chan, H., Oballa, R., Huang, Z., Jones, 472

D.M., Ridsdale, A., Russell, R.S., Partridge, A.W., and Pezacki, J.P. 2014. Stearoyl-CoA 473

desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes. 474

Sci. Rep. 4: 4549. doi: 10.1038/srep04549. PMID: 25008545. 475

Mackenzie, J.M., Khromykh, A.A., and Parton, R.G. 2007. Cholesterol manipulation by West 476

Nile virus perturbs the cellular immune response. Cell Host Microbe 2(4): 229-239. doi: 477

10.1016/j.chom.2007.09.003. PMID: 18005741. 478

Mazièrel, J.C., Landureau, J.C., Giral, P., Auclair, M., Fall, L., Lachgar, A., Achour, A., and 479

Zagury, D. 1994. Lovastatin inhibits HIV-1 expression in H9 human T lymphocytes cultured 480

in cholesterol-poor medium. Biomed. Pharmacother. 48(2): 63-67. doi: 10.1016/0753-481

3322(94)90077-9. PMID: 7522603. 482

Mboko, W.P., Mounce, B.C., Emmer, J., Darrah, E., Patel, S.B., and Tarakanova, V.L. 2014. 483

Interferon regulatory factor 1 restricts gammaherpesvirus replication in primary immune 484

cells. J. Virol. 88(12): 6993-7004. doi: 10.1128/jvi.00638-14. PMID: 24719409. 485

Mesmin, B., Bigay, J.l., Moser von Filseck, J., Lacas-Gervais, S., Drin, G., and Antonny, B. 486

2013. A four step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the 487

Page 22 of 33



Draft

ER-Golgi tether OSBP. Cell 155(4): 830-843. doi: 10.1016/j.cell.2013.09.056. PMID: 488

24209621. 489

Miller, S., and Krijnse-Locker, J. 2008. Modification of intracellular membrane structures for 490

virus replication. Nat. Rev. Microbiol. 6(5): 363-374. doi: 10.1038/nrmicro1890. PMID: 491

18414501. 492

Molina, S., Castet, V.r., Fournier-Wirth, C., Pichard-Garcia, L., Avner, R., Harats, D., 493

Roitelman, J., Barbaras, R., Graber, P., Ghersa, P., Smolarsky, M., Funaro, A., Malavasi, F., 494

Larrey, D., Coste, J., Fabre, J.M., Sa-Cunha, A., and Maurel, P. 2007. The low-density 495

lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis 496

C virus. J. Hepatol. 46(3): 411-419. doi: 10.1016/j.jhep.2006.09.024. PMID: 17156886. 497

Moog, C., Aubertin, A.M., Kirn, A., and Luu, B. 1998. Oxysterols, but not cholesterol, inhibit 498

human immunodeficiency virus replication in vitro. Antivir. Chem. Chemother. 9(6): 491-499

496. PMID: 9865387. 500

Morris, L.L., Hartman, I.Z., Jun, D.J., Seemann, J., and DeBose-Boyd, R.A. 2014. Sequential 501

actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S 502

regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation 503

of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Biol. Chem. 289(27): 19053-504

19066. doi: 10.1074/jbc.M114.576652. PMID: 24860107. 505

Moser, T.S., Schieffer, D., and Cherry, S. 2012. AMP-activated kinase restricts Rift Valley fever 506

virus infection by inhibiting fatty acid synthesis. PLoS Pathog 8(4): e1002661. doi: 507

10.1371/journal.ppat.1002661. PMID: 22532801. 508

Munger, J., Bennett, B.D., Parikh, A., Feng, X.J., McArdle, J., Rabitz, H.A., Shenk, T., and 509

Rabinowitz, J.D. 2008. Systems-level metabolic flux profiling identifies fatty acid synthesis 510

Page 23 of 33



Draft

as a target for antiviral therapy. Nat. Biotech. 26(10): 1179-1186. doi: 10.1038/nbt.1500. 511

PMID: 18820684. 512

Nguyen, U.T.T., Goody, R.S., and Alexandrov, K. 2010. Understanding and exploiting protein 513

prenyltransferases. ChemBioChem 11(9): 1194-1201. doi: 10.1002/cbic.200900727. PMID: 514

20432425. 515

Ogawa, S., Lozach, J., Benner, C., Pascual, G., Tangirala, R.K., Westin, S., Hoffmann, A., 516

Subramaniam, S., David, M., Rosenfeld, M.G., and Glass, C.K. 2005. Molecular 517

determinants of crosstalk between nuclear receptors and Toll-like receptors. Cell 122(5): 518

707-721. doi: 10.1016/j.cell.2005.06.029. PMID: 16143103. 519

Onoguchi, K., Yoneyama, M., Takemura, A., Akira, S., Taniguchi, T., Namiki, H., and Fujita, T. 520

2007. Viral infections activate types I and III interferon genes through a common 521

mechanism. J. Biol. Chem. 282(10): 7576-7581. doi: 10.1074/jbc.M608618200. PMID: 522

17204473. 523

Park, K., and Scott, A.L. 2010. Cholesterol 25-hydroxylase production by dendritic cells and 524

macrophages is regulated by type I interferons. J. Leukoc. Biol. 88(6): 1081-1087. doi: 525

10.1189/jlb.0610318. PMID: 20699362. 526

Peet, D.J., Turley, S.D., Ma, W., Janowski, B.A., Lobaccaro, J.M., Hammer, R.E., and 527

Mangelsdorf, D.J. 1998. Cholesterol and bile acid metabolism are impaired in mice lacking 528

the nuclear oxysterol receptor LXRα. Cell 93(5): 693-704. doi: 10.1016/S0092-529

8674(00)81432-4. PMID: 9630215. 530

Pezacki, J., Sagan, S., Tonary, A., Rouleau, Y., Belanger, S., Supekova, L., and Su, A. 2009. 531

Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte 532

Page 24 of 33



Draft

metabolism and the antiviral state it conveys against the hepatitis C virus. BMC Chem. Biol. 533

9(1): 2. doi: 10.1186/1472-6769-9-2. PMID: 19149867. 534

Radhakrishnan, A., Ikeda, Y., Kwon, H.J., Brown, M.S., and Goldstein, J.L. 2007. Sterol-535

regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block 536

transport by binding to Insig. Proc. Natl. Acad. Sci. U.S.A. 104(16): 6511-6518. doi: 537

10.1073/pnas.0700899104. PMID: 17428920. 538

Reboldi, A., Dang, E.V., McDonald, J.G., Liang, G., Russell, D.W., and Cyster, J.G. 2014. 25-539

Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I 540

interferon. Science 345(6197): 679-684. doi: 10.1126/science.1254790. PMID: 25104388. 541

Ren, S., Hylemon, P., Zhang, Z.-P., Rodriguez-Agudo, D., Marques, D., Li, X., Zhou, H., Gil, 542

G., and Pandak, W.M. 2006. Identification of a novel sulfonated oxysterol, 5-cholesten-543

3β,25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria. J. Lipid Res. 47(5): 1081-544

1090. doi: 10.1194/jlr.M600019-JLR200. PMID: 16505492. 545

Ren, S., and Ning, Y. 2014. Sulfation of 25-hydroxycholesterol regulates lipid metabolism, 546

inflammatory responses, and cell proliferation. Am. J. Physiol. Endocrinol. Metab. 306(2): 547

E123-E130. doi: 10.1152/ajpendo.00552.2013. PMID: 24302009. 548

Resh, M.D. 2006. Trafficking and signaling by fatty-acylated and prenylated proteins. Nat. 549

Chem. Biol. 2(11): 584-590. PMID: 17051234. 550

Richert, L., Castagna, M., Beck, J.-P., Rong, S., Luu, B., and Ourisson, G. 1984. Growth-rate-551

related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: 552

correlation with 3-hydroxy-3-methyl glutaryl CoA reductase activity. Biochem. Biophys. 553

Res. Comm. 120(1): 192-198. doi: 10.1016/0006-291X(84)91432-3. PMID: 6538790. 554

Page 25 of 33



Draft

Ridgway, N.D., Dawson, P.A., Ho, Y.K., Brown, M.S., and Goldstein, J.L. 1992. Translocation 555

of oxysterol binding protein to Golgi apparatus triggered by ligand binding. J. Cell Biol. 556

116(2): 307-319. doi: 10.1083/jcb.116.2.307. PMID: 1730758. 557

Roulin, P.S., Lötzerich, M., Torta, F., Tanner, L.B., van Kuppeveld, F.J., Wenk, M.R., and 558

Greber, U.F. 2014. Rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-559

current for the formation of replication compartments at the ER-Golgi Interface. Cell Host 560

Microbe 16(5): 677-690. doi: 10.1016/j.chom.2014.10.003. PMID: 25525797. 561

Rydberg, E.K., Salomonsson, L., Hultén, L.M., Norén, K., Bondjers, G.r., Wiklund, O., 562

Björnheden, T., and Ohlsson, B.G. 2003. Hypoxia increases 25-hydroxycholesterol-induced 563

interleukin-8 protein secretion in human macrophages. Atherosclerosis 170(2): 245-252. doi: 564

10.1016/S0021-9150(03)00302-2. PMID: 14612204. 565

Sagan, S.M., Rouleau, Y., Leggiadro, C., Supekova, L., Schultz, P.G., Su, A.I., and Pezacki, J.P. 566

2006. The influence of cholesterol and lipid metabolism on host cell structure and hepatitis 567

C virus replication. Biochem. Cell Biol. 84(1): 67-79. doi: 10.1139/o05-149. PMID: 568

16462891. 569

Schoggins, J.W., and Randall, G. 2013. Lipids in innate antiviral defense. Cell Host Microbe 570

14(4): 379-385. doi: 10.1016/j.chom.2013.09.010. PMID: 24139397. 571

Shibata, N., Carlin, A.F., Spann, N.J., Saijo, K., Morello, C.S., McDonald, J.G., Romanoski, 572

C.E., Maurya, M.R., Kaikkonen, M.U., Lam, M.T., Crotti, A., Reichart, D., Fox, J.N., 573

Quehenberger, O., Raetz, C.R.H., Sullards, M.C., Murphy, R.C., Merrill, A.H., Brown, 574

H.A., Dennis, E.A., Fahy, E., Subramaniam, S., Cavener, D.R., Spector, D.H., Russell, 575

D.W., and Glass, C.K. 2014. 25-hydroxycholesterol activates the integrated stress response 576

Page 26 of 33



Draft

to reprogram transcription and translation in macrophages. J. Biol. Chem. 288(50): 35812-577

35823. doi: 10.1074/jbc.M113.519637. PMID: 24189069. 578

Song, B.L., Sever, N., and DeBose-Boyd, R.A. 2005. Gp78, a membrane-anchored ubiquitin 579

ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of 580

HMG CoA reductase. Mol. Cell 19(6): 829-840. doi: 10.1016/j.molcel.2005.08.009. PMID: 581

16168377. 582

St. Vincent, M.R., Colpitts, C.C., Ustinov, A.V., Muqadas, M., Joyce, M.A., Barsby, N.L., 583

Epand, R.F., Epand, R.M., Khramyshev, S.A., Valueva, O.A., Korshun, V.A., Tyrrell, D.L., 584

and Schang, L.M. 2010. Rigid amphipathic fusion inhibitors, small molecule antiviral 585

compounds against enveloped viruses. Proc. Natl. Acad. Sci. U.S.A. 107(40): 17339-17344. 586

doi: 10.1073/pnas.1010026107. PMID: 20823220. 587

Stehr, M., Elamin, A.A., and Singh, M. 2012. Cytosolic lipid inclusions formed during infection 588

by viral and bacterial pathogens. Microbes Infect. 14(13): 1227-1237. doi: 589

10.1016/j.micinf.2012.08.001. PMID: 22982567. 590

Strating, J.R., van der Linden, L., Albulescu, L., Bigay, J.l., Arita, M., Delang, L., Leyssen, P., 591

van der Schaar, H.M., Lanke, K.H., Thibaut, H.J., Ulferts, R., Drin, G., Schlinck, N., 592

Wubbolts, R.W., Sever, N., Head, S.A., Liu, J.O., Beachy, P.A., De Matteis, M.A., Shair, 593

M.D., Olkkonen, V.M., Neyts, J., and van Kuppeveld, F. J. 2015. Itraconazole inhibits 594

enterovirus replication by targeting the oxysterol-binding protein. Cell Rep. 10(4): 600-615. 595

doi: 10.1016/j.celrep.2014.12.054. PMID: 25640182. 596

Su, A.I., Pezacki, J.P., Wodicka, L., Brideau, A.D., Supekova, L., Thimme, R., Wieland, S., 597

Bukh, J., Purcell, R.H., Schultz, P.G., and Chisari, F.V. 2002. Genomic analysis of the host 598

Page 27 of 33



Draft

response to hepatitis C virus infection. Proc. Natl. Acad. Sci. U.S.A. 99(24): 15669-15674. 599

doi: 10.1073/pnas.202608199. PMID: 12441396. 600

von Euler, U.S. 1936. On the specific vaso-dilating and plain muscle stimulating substances from 601

accessory genital glands in man and certain animals (prostaglandin and vesiglandin). J. 602

Physiol. 88(2): 213-234. doi: 10.1113/jphysiol.1936.sp003433. PMID: 16994817. 603

Walther, T.C., and Farese, R.V. 2012. Lipid droplets and cellular lipid metabolism. Annu. Rev. 604

Biochem. 81(1): 687-714. doi: 10.1146/annurev-biochem-061009-102430. PMID: 605

22524315. 606

Wang, C., Gale Jr, M., Keller, B.C., Huang, H., Brown, M.S., Goldstein, J.L., and Ye, J. 2005. 607

Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus 608

RNA replication. Mol. Cell 18(4): 425-434. doi: 10.1016/j.molcel.2005.04.004. PMID: 609

15893726. 610

Wang, H., Perry, J.W., Lauring, A.S., Neddermann, P., De Francesco, R., and Tai, A.W. 2014. 611

Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV 612

replication membrane integrity and cholesterol trafficking. Gastroenterology 146(5): 1373-613

1385.e1311. doi: 10.1053/j.gastro.2014.02.002. PMID: 15893726. 614

Wymann, M.P., and Schneiter, R. 2008. Lipid signalling in disease. Nat. Rev.Mol. Cell. Biol. 615

9(2): 162-176. doi: 10.1038/nrm2335. PMID: 18216772. 616

Zelcer, N., Sharpe, L.J., Loregger, A., Kristiana, I., Cook, E.C., Phan, L., Stevenson, J., and 617

Brown, A.J. The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and 618

affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis 619

pathway. Mol. Cell. Biol. 34(7): 1262-1270. doi: 10.1128/mcb.01140-13. PMID: 24449766. 620

621

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Table 1: Summary of viruses responding to 25HC treatment 622

Virus Reference

Ebola Virus (Liu et al. 2013)

Gammaherpesvirus (Mboko et al. 2014)

Hepatitis C virus (Chen et al. ; Pezacki et al. 2009; Sagan et al.

2006; Su et al. 2002)

Herpes Simplex Virus-1 (Blanc et al. 2013; Liu et al. 2013)

Human immunodeficiency virus (Liu et al. 2013; Moog et al. 1998)

Human papillomavirus-16 (Civra et al. 2014)

Human rotavirus (Civra et al. 2014)

Influenza A virus (H1N1) (Blanc et al. 2013)

Mouse cytomegalovirus (Blanc et al. 2013)

Murine gamma herpes virus 68 (Blanc et al. 2013; Liu et al. 2013)

Nipah virus (Liu et al. 2013)

Poliovirus (Arita et al. 2013)

Rhinovirus (Civra et al. ; Hofer et al. 1994; Roulin et al.)

Rift Valley Fever Virus (Liu et al. 2013)

Russian Spring-Summer Encephalitis

Virus

(Liu et al. 2013)

Varicellar Zoster Virus (Blanc et al. 2013)

Vesicular stomatitis virus (Liu et al. 2013)

West Nile virus (Mackenzie et al. 2007)

623

624

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Figure 1: Summary of 25HC’s effects on sterol homeostasis. An overview of 25HC’s 625

multiple modes of action in the sterol pathway. (A,B) In low sterol and oxysterol conditions, the 626

SREBP/SCAP complex is transported to the Golgi, where SREBP is cleavage into its mature 627

from cleavage by two proteases (S1P and S2P). (C) This mature form translocates to the 628

nucleus, where it regulates genes associated with lipid metabolism. (D) In sterol- and oxysterol- 629

rich conditions, cholesterol binds to SCAP, or 25HC binds to INSIG, inducing conformational 630

changes which promote binding between SCAP and INSIG, retaining the SREBP/SCAP 631

complex at the ER, preventing SREBP activation. (E) Lastly, high oxysterol levels acts as an 632

agonist for LXR signalling, which regulates genes associated with cholesterol efflux and bile 633

synthesis. (F) Lastly, 25HC also promotes association of INSIGs with HMGCR, the enzyme 634

catalyzing the rate limiting step of cholesterol biosynthesis, and promotes its degradation via the 635

ubiquitin-proteasome system. 636

Figure 2: Overview of 25HC’s antiviral effects on HCV life cycle. IFN-stimulated 25HC 637

production has the potential to impair HCV infection at several stages of its viral life cycle. An 638

overview of the HCV lifecycle is shown in the top left image. 25-HC has the potential to 639

influence the virus’ life cycle at three main stages: entry and fusion, replication, and assembly. 640

(A) At the stage of entry, 25HC decreases SREBP-regulated LDLR expression, a proposed entry 641

receptor for the virus (Molina et al. 2007). Also, 25HC modulates the lipid composition of the 642

plasma membrane, potentially modulating fluidity and impairing subsequent viral fusion. (B) 643

Similarly, modulation of cellular membranes will influence HCV replication complex formation, 644

which relies on significant ER membrane remodeling. The virus also depends on OSBP proteins 645

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as a key host factor for viral replication and virion maturation (Amako et al. 2009; Wang et al.). 646

25HC’s interaction with OSBP could interfere with the virus’ shuttling of cholesterol to 647

replication complexes. HCV hijacks the geranylgeranylated host factor FBL2 for replication; 648

however, this may be impaired due to 25HC’s effects on protein prenylation. The virus also 649

relies on lipid droplets as a platform for assembly. There should be an overall decrease in 650

cellular lipid content (and lipid droplets) due to 25HC’s inhibition of the SREBP signalling. (C) 651

25HC should also exert broad antiviral effects due to potentiation of IFN signalling and 652

activation of the integrated stress response. 653

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Canadian Society of Microbiologists’ Armand -Frappier Gold ... › bitstream › 1807 › ... · 41 as potent modulators of cellular metabolism (Brown and Goldstein 1974; Kandutsch