Oxysterol binding protein (OSBP) binds 25-hydroxycholesterol (25OH) and cholesterol, which regulates PH and FFAT domain interaction with the Golgi apparatus and endoplasmic reticulum, respectively. Adjacent to these domains is a phosphorylated serine-rich motif (SRM, T379, S381, S384, S387, S388, S391) that we hypothesize controls sterol transport by OSBP. To test this, OSBP dephospho-mimics or phospho-mimics were expressed in CHO cells. Western blot analysis showed that the S381 is phosphorylated by PKA and is required for phosphorylation of down-stream serine residues. When expressed in OSBP-null CHO cells, there was no difference in the localization of the OSBP mutants, and all mutants restored SM synthesis in response to 25OH. Recombinant OSBP 5S?5E had increased cholesterol binding and extraction, and decreased cholesterol transfer to liposomes compared to OSBP. OSBP 5S?5E also bound VAP more efficiently. A model is proposed wherein SRM phosphorylation facilitates VAP association with the ER and increases cholesterol extraction.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.

A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.

7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA 2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [ 3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of CPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388B1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA 2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.

Oxisteróis (por exemplo, 7hidroxicolesterol) são gerados por modificações oxidativas que ocorrem na molécula de colesterol. Podem ser encontrados em elevados níveis plasmáticos em pacientes com aterosclerose e como componentes da placa aterosclerótica. Considerando que o colesterol é o principal componente da cavéola (domínios específicos da membrana plasmática que ancoram diversas proteínas de sinalização) formulamos a hipótese que os oxisteróis podem ser incorporados a estes domínios, interferindo com as vias de sinalização aí localizadas. Células endoteliais de veia umbilical humana (HUVECs) em cultura foram expostas a 7hidroxicolesterol (10g/mL) por diferentes tempos. Analisamos a incorporação desse oxisterol à cavéola utilizando espectrometria de massa e a atividade das proteínas de sinalização presentes neste domínio: óxido nítrico sintase endotelial (eNOS), CD40/CD40L, receptor do fator de crescimento de fibroblastos (rFGF), utilizando PCR quantitativo e imunoblots. Inicialmente mostramos que o 7hidroxycholesterol, em concentrações fisiológicas, foi incorporado às cavéolas mais acentuadamente que em outros domínios de membrana. Esse fenômeno impediu o desligamento entre eNOS e caveolina, prejudicando a função dessa enzima. Também mostramos que o receptor CD40 apresentou uma maior incorporação à cavéola e o rFGF manteve uma ativação mais longa quando células foram expostas ao 7hidroxicolesterol. Esses efeitos gerados pelo oxisterol não estavam relacionados à sua ação sobre mediadores inflamatórios ou receptores nucleares, desde que nenhuma diferença foi observada no perfil de citocinas ou na expressão de genes dependentes da ativação de LXR. Assim, concluímos que a incorporação de 7hidroxycholesterol nos domínios de cavéola pode interferir com vias de sinalização sabidamente envolvidas na aterogênese ou na ruptura da placa / Oxysterols (for example, 7hidroxycholesterol) are generated by oxidative modifications to cholesterol molecules. They have been described in high levels in patients with atherosclerosis and as components of the atherosclerotic plaque. Since cholesterol is the main component of caveolae (plasma membrane domains that anchor several signaling proteins), we hypothesized that oxysterol could be incorporated to these domains, interfering with the signaling networks that use this pathway. Human umbilical vein endothelial cells (HUVECs) in culture were exposed to 7hidroxycholesterol (10g/mL) for different times. We analyzed incorporation of this oxysterol to caveolae using mass spectroscopy and the activity of signaling pathways present in these domains: endothelial nitric oxide synthase (eNOS), CD40/CD40L, fibroblast growth factor receptor (FGFr), using quantitative PCR and immunoblots. Initially we showed that 7hidroxycholesterol, in physiological concentrations, was incorporated to caveolae more prominently than to other plasma membrane domains. This phenomenon caused a difficulty in eNOS release from caveolin, impairing its function. We also showed that the receptor CD40 presented a stronger incorporation to caveolae and FGFr maintained a longer activation when cells were exposed to 7hidroxycholesterol. These oxysterol effects were not related to its action in inflammatory mediators or nuclear receptors, since no difference could be observed in cytokine profiles or in the expression of genes dependent on LXR activation. Therefore we conclude that 7hidroxycholesterol incorporation in caveolae domains may interfere with signaling pathways known to be involved in atherogenesis or in plaque rupture

Chronic exposure to some oxysterols might contribute to deterioration of human or environmental health. Oxysterols are both biomarkers of oxidative stress as well as mediators of its damage, and play a central role in many independent, but converging, disease processes, such as atherosclerosis, Alzheimer's disease, and age-related macular degeneration. Therefore, the aim of this thesis was to identify enzymes capable of transforming oxysterols to either reduce their toxicity or facilitate their metabolism or excretion. 7-ketocholesterol (7KC), being amongst the most cytotoxic and recalcitrant of these compounds, was the main focus of this work. We isolated various bacteria capable of utilizing 7KC as a sole carbon and energy source. One of these, Rhodococcus jostii RHAl , was subjected to rigorous transcriptomic and mutational analysis to elucidate its 7KC degradation pathway, which was similar, but not identical, to that of cholesterol. Metabolite screening revealed the reduction and subsequent removal of the 7-keto moiety prior to the step catalyzed by HsaC, the enzyme responsible for cleavage of sterol ring A. Furthermore, cloning and expression of a number of reductases from two gene clusters that were highly up-regulated during growth on 7KC identified three reductases that are active against several closely related structural analogs, though not 7KC itself. 7KC and a number of analogs were assayed for toxicity against human fibroblasts Several enzymes were overexpressed in these fibroblasts by transient transfection with mammalian expression vectors to screen for their ability to mitigate 7KC-induced cytotoxicity. A LAMP1/cholesterol oxidase chimera was found to be significantly cytoprotective to exposure to up to 50 μM 7KC compared to mock transfection as well as 7KC-transforming enzymes targeted to either the mitochondria or cytosol. Additionally, transfection with LAMP1 alone and treatment with 0.9% hydroxypropyl β-cyclodextrin also reduced toxicity. Therefore, it seems likely that addressing 7KC toxicity within the lysosome is critical for cytoprotection. This work provides preliminary evidence to support this approach, and may have implications for the treatment of oxysterol-associated diseases. However, further research is needed to evaluate the effects and safety of heterologous gene expression within the lysosome, both in vitro and in vivo.

Orientadores: Andrei Carvalho Sposito, Eliana Cotta de Faria / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:58:25Z (GMT). No. of bitstreams: 1 Virginio_VitorWilsondeMoura_M.pdf: 2355466 bytes, checksum: 77d04b1ff5936c26cd64f6d516447ff7 (MD5) Previous issue date: 2014 / Resumo: Atualmente aproximadamente 17 milhões de pessoas morrem por doenças cardiovasculares (DCV). No Brasil durante 2011 ocorreram cerca de 200 mil óbitos por DCV principalmente em decorrência da aterosclerose, classificada como uma doença imuno-inflamatória crônica que causa estreitamento luminal e gera diversas fatalidades como infarto agudo do miocárdio (IAM), acidente vascular encefálico (AVE), doença arterial periférica (DAP), entre outras. A DAP prediz outras DCV e é a principal causa de amputação devido a obstrução das artérias de membros inferiores. Análises histológicas indicam que a ruptura da placa, capa fibrosa do ateroma, erosão da placa e nódulo calcificado geram as principais fatalidades nas DCV. Até o momento não há biomarcadores plasmáticos para uso clínico afim de avaliar a estabilidade e a gravidade das placas ateroscleróticas, esse fato motiva a exploração de componentes envolvidos em doenças imuno-inflamatórias e no metabolismo lipídico tais como oxisteróis e marcadores da síntese e absorção do colesterol (MSAC). Os oxisteróis, principalmente 27-hidroxicolesterol (27-HDC) e 7-ketocolesterol estão envolvidos em mecanismos fisiopatológicos da aterosclerose e desempenham um papel na inflamação, citotoxicidade e necrose, além de funções no metabolismo lipídico. É relatado que os oxisteróis 24S- e 25-hidroxicolesterol (24S- e 25-HDC) estão envolvidos em doenças neurodegenerativas como Alzheimer. Estudos apontam uma causa em comum entre Alzheimer e aterosclerose, ambas são doenças inflamatórias crônicas, assim são necessárias mais investigações desses oxisteróis na doença aterosclerótica. A proteína de transferência de éster de colesterol (CETP) e proteína de transferência de fosfolípides (PLTP), assim como as lipoproteínas, podem mediar a transferência desses lípides. O objetivo desse trabalho foi quantificar concentrações de oxisteróis (24S-, 25-, 27-HDC e 7-ketocolesterol) e marcadores de síntese (desmosterol e latosterol) e absorção (?-sitosterol e campesterol) do colesterol em ateromas e plasma de 10 indivíduos com DAP (grupo DAP) e comparar com artérias sem ateroma de 13 voluntários (grupo CTL). Também relacionar esses esteróis com outros parâmetros como histórico clínico e proteínas de transferência de lípides. Foram realizadas análises clínicas e bioquímicas, classificações histológicas dos ateromas e quantificações de esteróis em artérias e plasma. Para interpretação dos dados obtidos foram usados testes estatísticos de comparação e correlação. Os dois grupos apresentaram uma similaridade nos dados clínicos e bioquímicos, diferenciando apenas em pressão arterial sistólica, atividade da PLTP, glicose e proteína C reativa (PCR), maiores no grupo DAP. A PCR se correlacionou positivamente com 24S-HDC, 25-HDC e 27-HDC plasmáticos. Comparação entre os grupos em relação a concentração de oxisteróis em artérias mostraram o 24S-HDC e o 27-HDC maiores no ateroma do grupo DAP, curiosamente o 25-HDC esteve maior em CTL. Quantificações de oxisteróis em plasma do grupo DAP mostrou um aumento do 25-HDC em relação ao grupo CTL. Não houve diferença significante para os MSAC. Os ateromas classificaram-se em 50% ruptura da placa, 20 % capa fibrosa do ateroma, 10% nódulo calcificado e 10% lesão fibrocálcica. Em conclusão mostramos o acumulo do 24S-HDC em placas de aterosclerose. O 27-HDC assim como outros estudos esteve em maior concentração nos ateromas indicando fortemente sua ação na patogênese dessa doença. Todos os oxisteróis sintetizados enzimaticamente dosados em plasma se correlacionaram positivamente com atividade inflamatória sistêmica, assim como 24S-HDC e 27-HDC em ateromas. Finalmente a atividade da PLTP esteve maior no grupo DAP / Abstract: Currently around 17 million people die from cardiovascular diseases (CVD). In Brazil, durant 2011 nearly 200.000 deaths occur mainly due atherosclerosis, classified as a chronic, immune-inflammatory disease that causes luminal narrowing and generates several outcomes as myocardial infarction, cerebrovascular accidents (stroke), peripheral arterial disease (PAD) and others. DAP predict other CVDs and is the leading cause of amputation due the clogged arteries in inferior members. Histological analysis indicates that plaque rupture, fibrous cap atheroma, plaque erosion and calcified nodules generate major CVD fatalities. So far, no plasma biomarkers for clinical use are available in order to evaluate the stability and extend of atherosclerotic plaques, therefore is necessary the investigation of the components involved in immune-inflammatory diseases and lipids metabolism such as oxysterols and cholesterol synthesis and absorption markers (CSAM). Oxidized sterols or oxysterols, manly 27-hidroxycholesterol and 7-ketocholesterol are involved in pathophysiological mechanisms of atherosclerosis and play a role in inflammation, apoptosis, necrosis and cytotoxicity, in addiction to lipid metabolism. Oxysterols 24S- and 25-hidroxycholesterol are involved with neurodegenerative diseases such Alzheimer¿s and resemble with atherosclerosis development since are chronical inflammatory diseases, for this reason is necessary an investigation of these oxysterols in atherosclerosis is needed. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) as well as lipoproteins, could mediate the transfer of the oxysterols. The aim of this work was quantified the levels of oxysterols (24S- 25-, 27- hidroxycholesterol and 7-ketocholesterol) and cholesterol synthesis (desmosterol and lathosterol) and absorption markers (?-sitosterol and campesterol) in atheroma and plaques from 10 individuals with PAD (PAD group) and compare with arteries without atheroma of 13 volunteers (CTL group). Also, relate these sterols with other parameters as clinical history, lipid transfer proteins and histological scores. Clinical and biochemical data were collected and sterols were quantified in arteries and plasma by gas chromatography coupled to a mass spectrophotometer (GCMS) and histological atheroma classification were determined. Comparison anlysis were performed using Mann-Whitney and correlations by Spearman¿s test. Both groups were not different regarding clinical and biochemical data, differencing only for systolic blood pressure, alcohol intake, PLTP activity, glucose and CRP, increased in PAD group. CRP was positively correlated with 24S-HDC, 25-HDC e 27-HDC from plasma. The comparisons of oxysterols levels in atheroma showed that 24-HDC and 27-HDC were increased in PAD group; interestingly, the 25-HDC was higher in CTL group. No significant differences were observed for CSAM between groups. The atheroma were classified as 50% plaque rupture, 20% fibrous cap, 10% calcified nodules and 10% of fibrocalcific injury. From the oxysterols from plasma of PAD group, only 25-HDC was increased compared to the CTL group. In conclusion, we demonstrated the accumulation of 24S-HDC in atherosclerotic plaques. The 27-HDC, as well as other studies, present higher levels in atheroma¿s, strongly indicating is role in the disease pathogenesis. All oxysterols measured in plasma were positively correlated with the systemic inflammatory activity. Finally, all atherosclerotic plaques were classified as lesions of advanced extension, according to the histological analysis / Mestrado / Clinica Medica / Mestre em Clinica Medica

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.