Advances in enzymatic oxyfunctionalization of aliphatic compounds

Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme can improve its stability and enable its reuse multiple times, resulting in better performance and commercial viability. Consequently, biocatalysis is being widely applied in the production of pharmaceuticals and some commodity chemicals. Moreover, its broader application will be further stimulated in the future by the emerging biobased economy.

Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified cholesterol-25-hydroxylase (CH25H) as a broadly antiviral ISG. CH25H converts cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). 25HC treatment in cultured cells broadly inhibited growth of enveloped viruses including VSV, HSV, HIV, and MHV68 and acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. It suppressed viral growth by blocking membrane fusion between virus and cell. In animal models, Ch25h-deficient mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and reversed T cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics. Copyright © 2013 Elsevier Inc. All rights reserved.

Introduction Metabolism and innate immunity are increasingly viewed as being connected and pathway mechanisms exist for sharing resources and cross-regulation (Im et al., 2011). Cholesterol is a vital component of cellular membranes and is critical to a broad range of cellular functions. As a result, elaborate homeostatic mechanisms have evolved to control sterol metabolism at multiple levels within and outside the cell (Horton et al., 2002). Importantly, several of these mechanisms involve 25-hydroxycholesterol (25HC). The synthesis of transcripts encoding virtually all enzymes of the sterol biosynthesis pathway is regulated by the transcription factor Srebp2. Moreover, the Srebp2 processing pathway, responsible for endoplasmic reticulum retention of the protein or proteolytic activation and translocation to the nucleus, is subject to inhibition by 25HC (Gong et al., 2006; Radhakrishnan et al., 2007; Yabe et al., 2002). In the mevalonate arm of the pathway, HMGCR serves as a key control point (Rodwell et al., 1976) and is further regulated by oxysterols (in particular 25HC) through proteasomal degradation elicited by INSIG1. In addition, 25HC may also regulate cholesterol homeostasis through its ability to activate liver X receptors (LXR) and a variety of other isoprenoid and sterol metabolic intermediates can negatively feedback on sterol biosynthesis (Espenshade and Hughes, 2007; Song et al., 2005). Viruses are known to alter cellular lipid metabolism to facilitate their own multiplication and, importantly, inhibition of cholesterol and fatty acid biosynthetic pathways has been shown to curtail virus replication, maturation and secretion (Clark et al., 2012; del Real et al., 2004; Munger et al., 2008; Peña and Harris, 2012; Potena et al., 2004; Spencer et al., 2011; Taylor et al., 2011; Ye et al., 2003; Yu et al., 2012). In this regard, interferon (IFN)-mediated downregulation of the sterol biosynthesis pathway has been shown to be an integral part of Mϕ host defense (Blanc et al., 2011; Watterson et al., 2012). It is well recognized that IFNs, in particular type I, play a pivotal role in inhibiting viral growth through the activation of IFN-stimulated genes (ISGs). However, the specific functions of many ISGs and the mechanisms by which they restrict viral replication are not well defined (O’Neill and Bowie, 2010). The notion that there is an intrinsic connection between the sterol network and IFN is further supported by studies showing IFN modulation of the cholesterol hydroxylase gene, Ch25h (Bauman et al., 2009; Diczfalusy et al., 2009; Park and Scott, 2010). Importantly, however, the precise mechanisms and physiological roles by which IFN regulates the sterol metabolic network remain unclear. Here, we have undertaken a comprehensive, unbiased investigation of the biological role of oxysterols produced by Mϕ during infection and upon IFN stimulation. We provide evidence for a biological role for 25HC as a lipid effector of the innate immune response, highlighting the targeting of host metabolic regulatory networks during viral infections and, more broadly, inflammation. Results Quantitative Oxysterol Profiling of Infected or Interferon-Activated Primary Macrophages We speculated that the synthesis of oxysterols might have a role in the negative regulation of sterol biosynthesis upon infection (Blanc et al., 2011). Accordingly, we performed a comprehensive quantitative determination of all known naturally occurring side-chain and steroid-ring oxysterol metabolites that are produced and/or secreted by either infected or interferon (β or γ) stimulated primary Mϕ (Figures 1A, 1C, and 1D). Bone-marrow-derived macrophages (BMDMs) were infected (mouse cytomegalovirus, MCMV) or stimulated (Ifn-β or Ifn-γ) for 12 hr and the oxysterol content of cells and medium measured by LC-MS(MSn) following derivatization (see Figure 1A for workflow schematic). By generating reconstructed ion chromatograms (RICs) for mono- and dihydroxycholesterols, epoxycholesterols, and mono- and dihydroxycholestenoic acids (see Figure 1B for chemical structure), essentially all naturally occurring oxysterols and their immediate downstream metabolites were profiled. Figure 1 shows the list for common oxysterols and those oxysterols not shown were inclusive for 3β-hydroxycholest-5-en-22-one, cholest-5-ene-3β,4β-diol, cholest-4-ene-3β,6α -diol, cholest-4-ene-3β,6β –diol, cholest-5-en-3β, 19-diol, cholest-5-ene-3β,22S-diol, cholest-5-ene-3β,24R-diol, cholest-5-ene-3β,7β,25-triol, cholest-(25R)-5-ene-3β,7β,26-triol, cholest-5-ene-3β,24,25-triol, 3β-hydroxycholest-(25S)-5-en-27-oic acid. The results of this oxysterol-metabolome and secretome screen are summarized in Figure 1C (intracellular) and 1D (secreted). Interestingly, in this study noticeable amounts of 25HC were found in unstimulated Mϕ but not media—a result that perhaps reflects a pre-existing subprimed activation of Mϕ (Kropp et al., 2011). Strikingly, amounts of only 25HC were markedly increased by infection or treatment with MCMV or Ifn (β or γ), respectively (Figures 1C and 1D). In this study, the only other detectable oxysterols (with systemic names provided in brackets) were 24(S),25-epoxycholesterol (24S,25-epoxycholest-5-en-3β-ol), 3β,7α-dihydroxycholestenoic (3β,7α-dihydroxycholest-[25R]-5-en-26-oic) acid and 7α,25-dihydroxycholesterol (cholesten-5-en-3β,7α,25-diol) but at extremely low levels. Because LC-MS(MSn) assays are exceptionally sensitive and these products were detected in the media only, it is possible that they were generated by auto-oxidation. Notably, no other oxysterol or downstream 3-oxo-4-ene metabolites of the listed oxysterols were found. Similarly, we found no evidence for oxysterol mono- or disulphates. The sulphation of oxysterols is known to catabolically inactivate oxysterol activity and also generate alternative-biologically active products. However, we find no detectable presence of either the 3- or 25-monosulphate or 3,25-disulphate. To avoid potential issues relating to serum-derived lipid contamination, we proceeded to investigate the production of 25HC using lipid-depleted media. Time course experiments revealed that the majority of 25HC was secreted during the first 8 hr of incubation, and levels continued to rise more gradually until the 24 hr endpoint (Figures 2A and 2B). As previously reported for cells grown in normal media, a steady and significant fall in free cholesterol, and its direct precursor desmosterol, was observed with time suggesting a reduced biosynthetic output (Figure 2C; data not shown) (Watterson et al., 2012). In summary, the LC-MS(MSn) experiments revealed 25HC as the only oxysterol notably upregulated following macrophage infection or activation by IFNs. This occurred during the first hours after infection or treatment before overall levels of cholesterol in treated cells fell notably below those of the control. Figures 2D and 2E show data that suggests that the highly-specific increase in 25HC was attributable to upregulated expression of the hydroxylase Ch25h; levels of the only other expressed hydroxylases Cyp27a and Cyp7a that are also implicated in 25HC metabolism were unaltered. When a different perspective on this result was sought and interferon or polyI:C was used to stimulate BMDMs, increased levels of secreted 25HC were clearly detectable 4 hr after treatment. PolyI:C is a potent Tlr3 agonist that stimulates high levels of Ifn-β production (Figure 2F). Notably, in contrast to the observed decline in Ch25h transcript expression, levels of secreted 25HC metabolite levels remained relatively high until 24 hr in these experiments. We conclude from the above data that interferon activation or MCMV infection of Mϕ results in a large and highly focused production of intracellular and secreted 25HC. Exogenous Treatment and Endogenous Secretion of 25HC by BMDM Has Broad Antiviral Potency It is possible that 25HC might function as an antiviral mediator in the IFN-regulated sterol metabolic network (Blanc et al., 2011). To test this idea, we sought to address whether physiologically relevant concentrations of 25HC, secreted by activated Mϕ, are capable of conferring an antiviral state. We first determined in dose-response experiments the inhibitory concentrations (IC) for viral growth of MCMV in normal serum (i.e., sterol-replete media would favor restricted Srebp2 processing). Figure 3A shows that under these conditions 25HC has an IC10 of approximately 0.1 μM and an IC50 of 2 μM and did not induce a cytotoxic effect (see Figure S1 available online). Because this level of inhibition is greater than the known levels of 25HC that are sufficient to disrupt Srebp processing (Radhakrishnan et al., 2007), we infer from these experiments that in sterol-replete conditions 25HC is unlikely to involve the Srebp processing pathway for maximal antiviral activity (Adams et al., 2004; Yabe et al., 2002). Because cells grown under lipid-deficient media conditions have an increased dependency on sterol biosynthesis, we also evaluated the antiviral effect of 25HC under lipid-depleted conditions (Sun et al., 2007). As shown in Figures 3A and 3B, there is an increase in sensitivity of MCMV to inhibition by 25HC under these conditions. This antiviral effect is specific to 25HC because other related enzymatically generated oxysterols, 19-HC and 7α-HC, fail to repress viral infection (Figure 3B). These oxysterols are known not to inhibit the Srebp-pathway (Radhakrishnan et al., 2007). In contrast, other oxysterols known to inhibit the Srebp-pathway through INSIG, such as 27-HC and 24(S), 25 epoxycholesterol, are similarly capable of inhibiting MCMV growth (Figure S1B). These observations point to the possibility that although the Srebp pathway is not absolutely essential for mediating the antiviral effects of 25HC, it might contribute toward enhancing the sensitivity to 25HC inhibition. The physiological relevance for the lipid dependency is presently unclear. The differential sensitivities under sterol-replete and -depleted conditions raise the possibility for multiple levels of control. To determine whether the differential modes of inhibition by 25HC are specific to MCMV or whether it might have a broader antiviral role, we next evaluated, in normal or lipid-depleted conditions, its potency to inhibit a range of other viruses in different cell systems. Figures 3C–3F show the quantitative determination of the dose-dependent potency of 25HC to inhibit Influenza A (H1N1), herpes simplex virus-1 (HSV-1), varicellar zoster virus (VZV) and murine gamma herpes virus 68 (MHV-68) viral infections in MDCK, HeLa, MeWo, and BHK cells, respectively. The results of these titration experiments clearly show a broad and highly potent (IC50 values ranging from 20 nM to 750 nM) antiviral role of 25HC (Figures 3D–3F). This effect is selective because Figure S1C shows that under the conditions tested, 25HC does not specifically inhibit Adenovirus 5 or 19a. We conclude that 25HC has a high potency to inhibit a broad spectrum of viruses from high to low physiological concentrations depending on lipid conditions and virus-host cell system. The pharmacologic agent LY295427 (3α,4α,5α)-4-(2-propenylcholestan-3-ol) is a known antagonist of 25HC, whereas LY306039, the 3β-isomer of LY295427, fails to derepress the effect of 25HC (Janowski et al., 2001). We used these agents to test whether they have any efficacy in interfering with the antiviral activity mediated by 25HC. Figure 4A shows the ability of exogenous 25HC to potently inhibit viral growth and that the observed antiviral effect can be completely blocked in the presence of LY295427 but not LY306039. These results validated our subsequent use of LY295427 in interfering with endogenously secreted 25HC. Accordingly, conditioned media (10 hr posttreatment) from mock-treated, infected, Ifn-β, or Ifnγ-treated Mϕ was incubated with vehicle alone, or with LY295427 or LY306039 and then applied to MCMV-infected NIH 3T3 cells. As expected, mock-conditioned media failed to inhibit viral growth and drug treatment had no effect (Figure 4B). Meanwhile, conditioned media from infected or IFN-treated cells had pronounced antiviral activity. In the presence of LY295427, antiviral activity was partially blocked (Figure 4B). We interpret the results of these experiments to show that endogenously secreted 25HC has a potent antiviral activity and, if we assume, LY295427 is an exclusive inhibitor of 25HC, this accounts for at least 40%–50% of the antiviral secretome activity. It is likely that the secretion of other antiviral cytokines rather than an incomplete blockade provides the remaining antiviral activity. Overall, these experiments demonstrate that Mϕ activated Ch25h generates biological antiviral activity through paracrine, and in the case of Mϕ also autocrine effects of 25HC. 25HC Antiviral Actions Are LXR Independent and Enantioselective The above studies raise the possibility for potentially direct and indirect modes of 25HC action. It is conceivable that inhibitory effects of 25HC above 0.5 μM might involve an indirect membrane-mediated mechanism, whereas those below 0.5 μM may have a direct protein mediated mode of action. It is also plausible that 25HC could be metabolized to a more potent antiviral metabolite. A candidate pathway is the Epstein-Barr virus-induced gene 2 (EBI2/GPR183) enzyme system (Hannedouche et al., 2011; Liu et al., 2011). The natural ligand for EBI2 has been identified as 7α,25-diHC. In this pathway, a critical enzyme required for the generation of 7α,25-diHC is CH25H, however, in our mouse experiments Ebi2 gene expression is downregulated in Mϕ upon productive and nonproductive infection as well as polyI:C treatment, in an IFN-dependent manner (Figure 4C), suggesting that it is unlikely to be involved in the antiviral response. Intriguingly, the coordinated IFN suppression of the Ebi2 mediated 25HC-catabolic-pathway further underscores the highly focused 25HC Mϕ response and is unlikely to involve an intermediary metabolite mode of action. To evaluate generalized membrane effects and further test the specificity of 25HC actions, we studied the activity of ent-25HC, an enantiomer of 25HC. In these experiments, we assumed the well-known ability of 25HC to interact and change the properties of cellular membranes would be enantiomer insensitive while protein specific inhibitory effects on viral growth would be restricted to 25HC. Figure 4D shows that ent-25HC has antiviral activity but only at concentrations exceeding 1 μM, which is below the nonselective effects of oxysterols on viral infection and is an order of magnitude less potent at inhibiting MCMV than 25HC (Figure 4D). These results show that the effect of 25HC on antiviral activity is highly enantio-selective, consistent with the idea that 25HC antiviral functions occurs through a specific protein target(s). Because ent-25HC can exert an antiviral effect at high concentrations it is likely that this activity occurs via an indirect mode of action mediated at the membrane level. Notably, the antiviral effect of ent-25HC provides evidence for Srebp-independent antiviral pathway that might act as a cofactor for membrane proteins or acting as a determinant for membrane lipid composition or compartmentalization (Contreras et al., 2012). In the case of lipid-depleted conditions, our data is consistent with a subordinate inhibition of the Srebp pathway by 25HC and by other oxysterols such as 24S, 25-epoxycholesterol or 27-HC. Notably, these oxysterols are also known to activate LXR (Janowski et al., 2001). Thus, we next evaluated the transcriptional responses of LXR and Srebp target genes in Ifnγ-treated or MCMV-infected Mϕ. In these experiments, molecular profiling of Ifnγ and MCMV infected Mϕ every 30 min for 12 hr showed the expected downregulation of Srebp2 target messenger RNAs (mRNAs) including Insig1 (Figure 4E). However, the temporal dynamic changes were complex. In this analysis, increased expression of Ch25h mRNA levels is associated with a subsequent activation of the LXR target genes Mylip and Abcg1. To directly test whether LXR ligand activation is sufficient to develop an antiviral state, we treated MCMV infected cells with 1 μM of the potent synthetic LXR ligands (GW3965 and T0901317). Although these ligands were active in LXR-dependent reporter assays (Figure S2A), they failed to elicit an antiviral response and, therefore, we conclude that LXR activation is insufficient to affect viral inhibition of MCMV in both normal or lipid depleted media conditions (Figures 4G and 4H; Figures S2B and S2C). 25HC Antiviral Action Partially Involves the Mevalonate Branch, Blocking Postentry Viral Growth A decrease in the Srebp2 target genes of the sterol pathway and especially the mevalonate-isoprenoid side branch is notable (in particular Mvd and Fdps) (Figures 4E and 4F). Mevalonate is a shared precursor of coenzyme Q, ubiquinone, dolichol, farnesyl, and geranylgeranyl pyrophosphates. The latter two isoprenoids are necessary for protein prenylation. Many viruses require prenylation of essential viral or host proteins (e.g., the large delta antigen of HDV or the pseudorabies virus tegument protein) (Bordier et al., 2003; Clase et al., 2003) or (e.g., host protein FBL2 for hepatitis C virus [HCV] or RhoA activation for RSV) (Ye et al., 2003) (Wang et al., 2005) (Gower and Graham, 2001). In the case of MCMV, we find, upon inhibition of the prenyltransferase branch by RNAi, that viral replication is selectively reduced upon knockdown of GGTase II but not GGTase I or farnesyltransferase (Figure 4J), confirming previous work defining the dependency of MCMV for the isoprenoid pathway (Blanc et al., 2011). In the present study, Rabggta and Rabggtb small interfering RNA (siRNA) reduced the abundance of their target transcript by around 60% (Figure S4F) and none of the above siRNA significantly reduced cell viability (Figure S4D). To investigate whether 25HC antiviral functions involve the mevalonate branch, we treated MCMV infected cells with nonsaturating inhibitory levels of 25HC ( 5 μM), viral growth is not recovered with MEV, GGOH, or FOH cotreatment (data not shown) indicating that under these conditions the isoprenoid-prenylation arm is not required. It is possible that other derivatives such as dolichol may be required. Altogether, these investigations are indicative that for MCMV, 25HC blocking of the mevalonate branch imparts a contributory role but is not absolutely essential. As expected, inhibitors of GGTase I (GGTT-2133) or farnesyltransferases (FTI-277) fail to inhibit MCMV, whereas psoromic acid (an inhibitor of GGTase II [Deraeve et al., 2012]) restricts viral growth (Figure 4K). None of these compounds significantly affected cell viability (Figure S2E). We next investigated at what stage in infection 25HC blocks MCMV growth. The results shown in Figure 5A indicate that the level of internalized MCMV genomes is unaffected by 25HC. Furthermore, in plaque reduction assays the number of primary infectious foci is equivalent in the presence or absence of 25HC (Figure 5E). These results argue against viral entry as a primary mode for MCMV inhibition. In this connection, we also find that 25HC is highly effective at blocking (at 40 nM inhibitory concentration) VZV infection of MeWo cells (Figure 3E). This represents an infection model in which cell-free virus is not produced and the infection process is exclusively dependent on cell-cell spread. 25HC at high concentrations has been shown to promote apoptosis, a well-characterized antiviral mechanism, in a range of cell types including Mϕ (Rusiñol et al., 2004). Notably, however, as shown for NIH 3T3 cells (Figure 5B), 25HC at high antiviral concentrations (10 μM) fails to significantly induce cellular apoptosis in comparison with staurosporine. Again, we cannot rule out that other cell-virus systems may employ a 25HC mediated apoptotic antiviral response. In this regard, we have found BMDM to be especially sensitive to 25HC concentrations above 2 μM. We noted marked inhibitory effects on plaque development for all viruses investigated (Figure 5C–5G). Dose response analyses of MCMV plaque formation clearly indicated that the ability of the virus to spread decreased with increasing dose of 25HC (Figures 5C and 5D), while no statistical differences in numbers of infection foci in the presence or absence of 25HC were obtained (Figure 5E). In plaque reduction assays a significant dose-dependent difference in the diameter of the viral plaque was observed (Figure 5E). Similar results were also obtained from plaque reduction assays for influenza virus (Figure 5F). A simple mathematical model that best fits the experimental data is the one in which growth dominates over susceptibility to infection (Figure 5H; Figure S3F; Movie S1). In agreement, single hit growth analyses of MCMV and influenza infection show that 25HC effectively inhibits viral growth (Figure 5G) and for MCMV, 25HC inhibits yields of intracellular and secreted virus to the same extent (Figure 5G). However, in the case of HSV-1 we observed a significant difference in both plaque size and number of plaques (Figure 5F; Figure S3E) suggesting viral entry and growth can be affected and further indicating the multiple levels of control exerted by 25HC depending on the virus-host cell system. In accordance with the above results we observed a reduction at the level of viral DNA replication by 25HC (Figure 5I). This is associated with reduced amounts of early (E1) and late (M115) gene expression in a 25HC dose-dependent manner strongly supporting a postentry stage inhibition of MCMV infection (Figure S3A). For MCMV Mϕ infection, IFNs mediate antiviral effects through inhibition of the major immediate-early promoter (MIEP) (Kropp et al., 2011). We also find that 25HC imparts, in a dose dependent manner, inhibition of the MIEP monitored by using either reporter virus assay or the endogenous ie1 expression levels, resulting in approximately 50% inhibitory activity in comparison with Ifnγ treatment (Figure 5J; Figures S3B and S3C). Interferon Stimulation of 25HC Production Involves Direct Binding of Stat1 to Ch25h Promoter A central question regarding the role of 25HC in terms of innate immunity is how IFN signaling precisely regulates Ch25h. Thus, in our next experiments we sought to investigate in further detail the regulatory interferon dependence of the 25HC responses by using host and viral genetic knockouts in our macrophage-infection experiments. First we assessed whether the IFN-β plays a role in mediating the induction 25HC in infection. Figure 6A shows that greater than 80% of the induction response of Ch25h is lost in Ifnb1 −/− Mϕ, whereas 100% of the response is eliminated in Ifnar1 −/− Mϕ. Because all type I IFNs signal through the Ifnar1 receptor, these results indicate that upon infection of Mϕ, Ifn-β is the primary mediator, whereas other members of the type 1 interferon family serve a minor nonredundant role. To determine whether a productive infection is required, we used a mutant virus (MCMVdie3) that is fully proficient in infecting cells but fails to initiate viral gene expression. In the following experiments, infection of Mϕ with MCMVdie3 resulted in an equal response of Ch25h activation in comparison with the parental wild-type (WT) virus but with slightly delayed temporal kinetics (Figure 6B). In this case, and in comparison with Ifnar1 −/− -infected cells, Ifn-β dominates the response (Figure 6B). These results show that productive infection is not required; indicating that pattern recognition receptor activation by the viral particle is sufficient to activate the transcriptional induction of Ch25h. In our Mϕ cultures, Ifn-γ is not expressed but, as shown in Figure 1, is a potent inducer of Ch25h activity. Figure 6C shows that infection or treatment with exogenous Ifn-β of Ifnar1 −/− Mϕ abolishes the induced levels of 25HC, whereas exogenous Ifn-γ treatment does not. Because signal transducer and activator of transcription factor 1 (Stat1) is activated by Ifn-γ though its receptor Ifngr and Stat1 and Stat2 are activated by Ifn-β through its receptor Ifnar1, we next tested the ability of MCMV, Ifn-β and Ifn-γ to activate Ch25h mRNA levels in Stat1 −/− cells. Figure 6D clearly shows that genetic elimination of Stat1 results in the marked loss of the ability of Mϕ to activate Ch25h upon infection or treatment with type 1 or type 2 IFNs. To further investigate the kinetic transcriptional induction of Ch25h, we quantitatively determined the de novo synthesis of RNA using 4-thiouridine incorporation. A schematic of this procedure is shown in Figure S4A. In these experiments, between 60 and 90 min after treatment, de novo synthesis of the Ch25h transcript increased by approximately 30-fold in Ifn-γ treated cells relative to the mock sample (Figure 6E). To test whether signaling from Ifnar1 was necessary for the induction of Ch25h during viral infection, we next conducted de novo synthesis experiments in Tyk2 −/− macrophage cells. The results shown in Figure 6F indicate that direct signaling by the receptor rather than an indirect or secondary mode of activation is crucial in virus-induced Ch25h synthesis and is consistent with the possibility of Stat1 involvement in this process. To explore this possibility further, we next conducted a microarray analysis of de novo transcription during the first 8 hr after IFN-γ stimulation of macrophages. An unbiased clustering analysis of temporal differential synthesis profiles identified a subset of 51 gene transcripts kinetically related to Ch25h, including Irf1 and Tap1, (Table S1; Figure 6G). Having demonstrated a notable increase in Ch25h transcript synthesis during the first 30 min after IFN-γ treatment, we sought to bioinformatically investigate promoter regions for common transcription factor binding sites. To this end, we applied a statistical analysis of transcription factor overrepresentation in transcripts increased by >2-fold during the first 30 min after IFN-γ treatment. An analysis of 109 transcripts identified (including Ch25h) (Table S2) revealed Stat1 as the only transcription factor identified with a combined Z score of >10 and Fisher score of 2-fold in the Ifn-γ-treated BMDM samples between 0 and 30 min were imported into the oPOSSUM software tool (http://www.cisreg.ca/cgi-bin/oPOSSUM/opossum) and analyzed as described in Supplemental Experimental Procedures. Isolation and Quantification of Viral Genome Copy Number by Using qPCR For DNA extraction, cells were extensively washed thrice 3 hr postinfection and total genomic DNA isolated and genome copy number normalized to input DNA or to gapdh using quantitative procedures described in Supplemental Experimental Procedures. Oxysterol Screening against MCMV, HSV-1, MHV-68, VZV, Influenza A, and Adenovirus For these experiments, respective host cell systems were conducted in black 96-well plates using media conditions and oxysterol treatment as described in the figure legends and Supplemental Experimental Procedures. The IC50 values were calculated as the concentration at 50% viral inhibition. Cell viability and toxicity assays were determined using the CellTiter Blue (CTB, Promega) reagent. Metabolite Treatment of Cells Murine embryo fibroblasts were infected with MCMV (MOI = 0.01). After adsorption, cells were washed 5 times with normal medium (DMEM, 10% FCS, L-Glutamine and Penicillin/Streptomycin). After washing, normal medium containing vehicle (Ethanol) or 25HC (1 μM) ± Geranylgeraniol (GGOH, 20 μM) (Sigma G3278) or Mevalonolactone (Mev, 20μM) (Sigma M4667), Farnesol (FOH, 20 μM) (Sigma F203) or Squalene (Sqle, 20 μM) (Sigma S3026) was added to the infected wells. After 4 days, supernatants were collected and MCMV titer calculated by plaque assay. Computational Prediction of Stat1 Binding Sites in the Ch25h Promoter To analyze and predict potential Stat1 binding sites in the promoter of Human and Mouse Ch25h genes, we used the open source software Toucan. In brief, 1Kb 5′ cis-regulatory regions upstream of the transcriptional start sites of human (ENSG00000138135) or mouse (ENSMUSG00000050370) Ch25h were imported into Toucan. Predicted transcription factor binding sites were then identified in these sequences using the MotifLocator algorithm. Position Weight Matrices for this analysis were derived from the TransFac database V7.0 (public) and the background model used was either mouse or human DBTSS promoters depending on input sequence. A default stringency threshold of 0.9 was used for all predictions. Chromatin Immunoprecipitation (ChIP) For ChIP of Stat1, 6 × 107 Mϕ were used per ChIP, as detailed in the Supplemental Experimental Procedures, including the analysis of ChIP-Seq data for UCSC Browser image of Ch25h locus.