No description available.

When grown in the presence of the sterol biosynthesis inhibitor, paclobutrazol, suspension cultures of Apium graveolens (celery) accumulate substantial amounts of I 4a-methylsterols, at the expense of 4-demethylsterols. These changes have been correlated with reduced synthesis of phosphatidylcholine (PC) via the CDP-base pathway (Roiph & Goad, 1991). It was subsequently proposed that changes in the membrane sterol composition of plant cells may regulate the activity of CTP: cholinephosphate cytidylyltransferase (CT), the rate-determining enzyme of this pathway (Kinney & Moore, 1989). In preliminary studies, the membrane-associated form of CT in A.graveolens, was found to exhibit optimal activity at pH 7.7, in the presence of 8.0 mM CTP and 3.5 mM Mg2t Microsomal membrane fractions, in which a large proportion of CT activity was found to reside, were analysed in terms of lipid composition. The predominant phospholipid in such membranes, PC, constituted approximately 70% of the total phospholipid content. Other, more minor constituents, included phosphatidylethanolamine (PE), phosphatidylglycerol (PU), phosphatidylinositol (P1), phosphatidylserine (PS), and phosphatidic acid (PA). All phospholipids present in A.graveolens were found to be rich in linoleate (18:2) and palmitate (16:0). Lesser amounts of stearate (18:0), oleate (18:1), and a-linolenate (a- 18:3), were also present. The major phytosterols in microsomes were identified as campesterol, stigmasterol, sitosterol, and isoflicosterol, with trace amounts of cholesterol and 24-methylene cholesterol. The sterol biosynthesis inhibitors, miconazole, terbinafine, fenpropimorph, and tomatidine, proved to be useflul tools in the manipulation of membrane sterol composition in suspension cultures of A.graveolens. Treatment with these inhibitors caused significant alterations in lipid composition with corresponding changes in the activity of membrane-associated CT. Terbinaflne and fenpropimorph caused a large increase in the stigmasterol/sitosterol ratio of cells with a concomitant stimulation of CT activity. The latter compound also resulted in the accumulation of various 9$, 19- cyclopropyl sterols. Similarly, the azasterol inhibitor, tomatidine, resulted in an enhancement of CT activity, but with very lift le change in the stigmasterol/sitosterol ratio of cells. Conversely, miconazole resulted in a decline in the stigmasterol/sitosterol ratio, corresponding to lower membrane-associated CT activity. The latter inhibitor also caused an accumulation of oleoyl residues in the PC fraction of cells, suggesting an inhibition of A 1 2-desaturase activity. Radiolabelling studies with [3IflS-adenosyl-L-methionine revealed a degree of coordinate regulation between the CDP-base and methyltransferase pathways of PC biosynthesis. Consequently, despite changes in CT activity, levels of phospholipid in most inhibitor-treated cultures remained relatively constant. Supplementation of miconazole-treated cultures with free fatty acids partially overcame the cytostatic nature of the azole inhibitor, with a concomitant reactivation of CT. Mono- and diunsaturated fatty acids were found to be the most effective compounds in this respect. The addition of stigmasterol or sitosterol to miconazoletreated cultures also resulted in partial growth restoration and reactivation of membrane-bound CT.

No description available.

Sterols play many important roles in physiology, including maintaining cell membrane integrity, and producing vitamin D and steroid hormones. Recent studies implicate sterol metabolism in the host innate immune response. Previous work, based on transcriptional profiling studies of mouse cytomegalovirus (MCMV) infection of primary bone-marrow-derived macrophages (BMDM, MΦ), uncovered a previously uncharacterized role of interferon in regulating the cholesterol pathway. Notably, Toll-like receptor (TLR) induced interferon modulates the suppression of SREBP2 (Sterol Regulatory Element-Binding Protein 2) activation, the master transcription factor for sterol biosynthesis. This finding resulted in the downregulation of the sterol biosynthesis pathway. However, how interferon is molecularly linked to sterol metabolism, and what part of the pathway mediates the antiviral effect remains unknown. The central hypothesis of the thesis is that the antiviral effect of interferon is in part mediated by secondary sterol metabolites and the dependency of viral replication on the host mevalonate branch of the sterol biosynthesis pathway. To test this hypothesis, my studies have examined the components of the host sterol pathway and their respective roles in influencing viral replication. Paradigmatically, I used MCMV and BMDM to explore the host- metabolic-virus interactions. Specifically, my findings address the question of how MCMV replication depends on the sterol biosynthesis pathway, and how the pathway is modulated by interferon as an antiviral response. In Chapter 2, the importance of the sterol biosynthesis pathway for viral replication was investigated using a combination of gene silencing and pharmacological inhibitors. These studies demonstrated that resistance to viral infection through suppressing the cholesterol pathway is not due to a requirement of the virus for cholesterol itself, but instead involves the mevalonate-isoprenoid arm of the pathway. This branch of the pathway chemically links lipids to specific host proteins (protein prenylation). These results suggest a new role for the mevalonate arm during viral infection. In Chapter 3, I examined what part of the sterol pathway mediates the antiviral effects. Oxysterols are natural modulators of sterol biosynthesis, and are produced by the oxidation of cholesterol by the enzyme cholesterol hydroxylase. Oxysterol suppression of SREBP2 activation leads to transcriptional repression of the sterol biosynthesis pathway. Additionally, oxysterols also modulate cholesterol homeostasis through cholesterol efflux. My studies led to identifing cholesterol-25-hydroxylase (Ch25h) as an interferon-stimulated gene (ISG). CH25H oxidizes cholesterol to produce a soluble oxysterol metabolite, 25-hydroxycholesterol (25-HC). Treatment of cells with 25-HC resulted in antiviral effects against MCMV and MHV-68. 25-HC was found to have no effects on MCMV entry into the host cell, but rather mediated inhibition of viral gene transcription. In addition, 25-HC-specific antiviral effect partially involved the suppression of the isoprenoid pathway, rather than cholesterol efflux. This work uncovered a physiological role for 25-HC as a sterol-lipid effector of an innate immune pathway. The antiviral activity of 25-HC in a lipid replete condition was found to occur at a concentration higher than the concentration required to inhibit SREBP2 activation. This implies that the antiviral effects of 25-HC is independent of SREBP2 in sterol replete conditions. Conversely, the antiviral action of 25-HC was signifi enhanced in cells under sterol-depleted conditions, suggesting that the antiviral effect of 25- HC is likely mediated through multiple processes involving SREBP2 dependent and independent mechanisms. These sterol dependent and independent mechanisms are examined in Chapter 4, using pathway expression profiling and pharmacological synergy studies. These studies showed that 25-HC suppression of the isoprenoid synthetic pathway is crucial in controlling infection, but also highlighted that other 25-HC dependent antiviral mechanisms are likely to exist. The inhibition of the mevalonate-isoprenoid arm by statins and 25-HC clearly demonstrated that MCMV replication dependents on protein prenylation. Chapter 5 investigation showed that either chemical inhibition of geranylgeranylation of host proteins or limiting mevalonate production led to restriction of MCMV replication. Importantly, through a series of systematic loss of function siRNA screenings demonstrated that specific host RabGTPases mediating vesicular transport pathways play vital roles in the replication and the assembly of the virus. This finding provides new mechanistic insights in to the dependency of cytomegalovirus replication on the host cell trafficking pathways and lays the groundwork for further definition of this important aspect of host-viral interactions. In summary, the overall findings of this research support the original hypothesis, by highlighting the importance of the host mevalonate-isoprenoid pathway, and provide further definition of the mechanisms and components linking sterol metabolism with interferon mediated antiviral effect.