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The oxysterol, 25-hydroxycholesterol (25-HC), is important for sterol metabolism and emerging evidence suggests that 25-HC plays a more critical role in immunity and infection. However, the precise antiviral mechanism and the target of 25- HC remains unclear. Here efforts were made to investigate the link between viral infection and the triggering of the 25-HC associated interferon (IFN) response, and how this dynamically alters the endogenous level of 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), a key enzyme that catalyses the production of the precursor of cholesterol and oxysterols. In this thesis I have sought to specifically explore the temporal changes and role of HMGCR in DNA virus (cytomegalovirus) and RNA (Influenza) virus infections. I hypothesise that HMGCR is a target for 25-HC associated IFN-mediated host defence against viral infection. To characterise HMGCR and test this hypothesis, the following objectives were defined: (1). To establish an experimental system to quantitatively study the endogenous HMGCR protein level; (2). To investigate the mechanism of the down-regulation of HMGCR involved in the IFN-mediated innate immune response; (3). To study the behaviour of HMGCR in the influenza virus induced 25-HC associated IFN-mediated innate immune response; (4). To study the behaviour of HMGCR in the cytomegalovirus induced 25-HC associated IFN-mediated innate immune response. Chapter 3, describes establishing an experimental system for the quantification of endogenous HMGCR levels. Different protein detection methods, including a modified western blot protocol and immunostaining, were tested. The results of RNA interference of HMGCR demonstrate that under lipid-deficient condition with the supplementation of mevastatin (an HMGCR inhibitor) the modified western blot protocol specifically detects endogenous HMGCR. This chapter lays the foundational work for the temporal analysis and testing the role of HMGCR in infection. In Chapter 4, the mechanism of the degradation of HMGCR following 25-HC and IFN treatments, in wild-type and Ch25h−/− mouse bone marrow derived macrophages (BMDMs), was investigated. Similar to 25-HC, IFN-γ treatment results in the drop of both the transcript and protein abundance of HMGCR in wild-type BMDMs. Differential temporal analysis of RNA and protein alterations and the use of proteasome inhibitors reveals that both 25-HC and IFN-γ lead to a marked reduction of HMGCR protein via a proteasomal degradation mechanism within early times of treatments. Further, the immediate reduction of HMGCR levels induced by IFN-γ was completely abrogated in Ch25h−/− BMDMs. Hence, the reduction of HMGCR following IFN-γ treatment is due to the de novo synthesis in macrophages of 25-HC. However, the decrease of Hmgcr gene expression was observed in not only wild-type but also Ch25h−/− BMDMs, suggesting additional mechanisms for regulating Hmgcr RNA levels. These results demonstrate the mechanism of the down-regulation of HMGCR resulted from the induction of IFN response during viral infection, is only partially due the de novo synthesis of 25-HC. In chapter 5, influenza A virus was used to investigate the role of HMGCR in the IFN-mediated innate immune response. The inhibition of HMGCR by RNA interference inhibited viral growth, suggesting the requirement of HMGCR for optimal intracellular viral growth. Viral infection in wild-type murine BMDMs reduced the endogenous HMGCR levels. However, the reduction of HMGCR at early times was prevented in Ch25h−/− BMDMs. Intriguingly, the decrease of HMGCR at late time points was still observed in Ch25h−/− BMDMs. These results indicate that the down-regulation of HMGCR with influenza virus infection in BMDMs at early times is completely due to the de novo synthesis of 25-HC; whereas at late times alternative pathways or mechanisms exist. Additionally, human epithelial A549 cells and A549/PIV5-V cells that are deficient in STAT1 were used to study the role of IFN pathway in the down-regulation of HMGCR at late times during viral infection. Results from these studies show that at late times the reduction of HMGCR is due to IFN-independent mechanisms. Chapter 6, extends these investigations to the herpes virus murine cytomegalovirus and infection of BMDMs. HMGCR is known to be essential for cytomegaloviral infections and 25-HC, statin and RNAi inhibition of HMGCR restrict viral growth. 25-HC is shown to reduce HMGCR at immediate early times of infection. However, most notably, the down-regulation of HMGCR was also observed in Ch25h−/− BMDMs at late times with murine cytomegalovirus infected BMDMs. These results confirm that alternative pathways or mechanisms exist, playing roles in the crosstalk between cholesterol metabolism and innate immune response. Collectively, this study characterises the role of HMGCR in the 25-HC associated IFN-mediated host defence against viral infection. Results indicate that, in addition to the IFN-mediated host response, alternative pathways or other mechanisms also result in the down-regulation of HMGCR during viral infection. HMGCR is at the crossroad of different pathways or mechanisms, and is therefore not only targeted by 25-HC. Hence, further questions can be addressed from these results: (1). What are the alternative pathways or mechanisms for the down-regulation of HMGCR? (2). How do these pathways or mechanisms work in hosts’ immune system? Answering these questions can contribute to refining the pathway map of innate immunity and understanding the precise role of HMGCR, or even the sterol biosynthesis pathway, in hosts’ immune response against pathogens.

The murine cytomegalovirus (MCMV) immediate early 1 (IE1) protein has been described as a trans-activator of viral and host gene expression. However, the precise role that IE1 plays in the viral life cycle, and in particular its effect on the host immune response is not known. This thesis investigates the functional relationship of the IE1 protein and the immune response induced after infection. By using an ie1-deletion mutant MCMV (MCMVdie1) it was demonstrated that, early after infection, tumor necrosis factor (tnf ) gene activation and protein production was significantly induced in infected-primary macrophages (M ) to a much greater extent than its wild type counterpart. In addition, preliminary studies on the signalling pathways activated upon infection were carried out in order to gain information about the pathways that might be involved in MCMVinduced modulation of tnf activation. Initial observations on the MAPK family members Erk1/2, p38 and JNK did not revealed any differential activation in the absence of IE1. However, due to a number of limitations, it was not possible to draw any firm conclusions from this study. Investigation of the role of IE1 in the in vivo production of TNF were also performed in both susceptible (BALB/c) and resistant (C57Bl/6) mice. These experiments confirmed the attenuated phenotype of MCMVdie1 in vivo, whereby the mutant strain grew to much lower titers than wild type. When cytokine production was assessed in relation to PFU levels a significant production of TNF after infection is observed in different organs of both mice strains. This raises the question whether IE1 contributes to MCMV modulation of TNF production in the natural host. Although, because it is still unclear whether the phenotype of MCMVdie1 in vivo is due to a defect in the virus or the result of a immune response, it was not possible to conclude unequivocally that IE1 is responsible for dampening this cytokine response. This thesis also tested whether the attenuated replication of MCMVdie1 in vivo was due to the increased TNF production induced after infection. An initial investigation in tnf depleted mice revealed that the MCMVdie1 growth phenotype is not due to TNF response. Overall, this study has provided insight into a potential immune modulatory function by MCMV associated with IE1 protein and the regulation of TNF in vivo and in vitro.

The betaherpesvirus Human Cytomegalovirus (HCMV) is estimated to be present in 40-80% of world population. HCMV infection in a healthy person causes mild symptoms, although the virus persists in the host in the latent form. Immunocompromised patients such as HIV and organ transplant patients as well as fetuses and neonatal babies with underdeveloped immune system are most affected by HCMV infection. CMVs are characterized by their strict species specifity; therefore humans are the only host for HCMV. The mouse cytomegalovirus (MCMV) has been the most useful animal model to explore HCMV spread and disease. CMVs encode G Protein-Coupled Receptors (GPCR) and these viral GPCRs are increasingly recognized as important regulators of pathogenesis and disease. HCMV for example encodes four of these GPCRs, whereas MCMV expresses two: M33 and M78. M33 shows constitutive signaling properties and activates various signaling pathways. M33 stimulates the PLC-&beta/PKC pathway via G&alphaq/11, however activation of NF-&kappaB and p38 MAP-kinase is independent of G-proteins. Importantly, M33 is essential for dissemination to or growth in salivary glands of immunocompetent mice and the G-protein signaling ability of M33 is necessary for viral growth at this site. Here we used the non-obese diabetic (NOD) and the immunocompromised NOD scid gamma (NSG) mouse models to assess a potential role for M33 as an immunomodulator and to further investigate the mechanism(s) by which M33 promotes viral growth in vivo. We are able to detect replication of &DeltaM33 viruses in the salivary glands of immunocompromised NSG mice; however it exhibited a 400x defect compared to wildtype MCMV. Since the growth phenotype is not completely reverted in the NSG mice, we conclude that M33 is not solely functioning to modulate the immune system. We also showed that M33 is dispensable for hematogenous dissemination within the host and that activation of G&alphaq signaling pathways alone is not sufficient to promote salivary gland growth as neither HCMV US28 nor a constitutively active form of G&alphaq are able to complement the unique functions of M33. We also describe here the generation and characterization of an in vitro cell culture model with cells extracted from mouse salivary glands. Specialized growth conditions resulted in cells that express markers of salivary gland epithelium, but none resulted in maintenance of epithelial markers similar to those expressed by the organ in vivo. Despite growth conditions that enable sustained expression of at least low levels of the salivary epithelial markers, M33 was not required for viral growth in these primary cells. Taken together, these studies suggest that M33 is necessary to allow viral growth within the three-dimensional structure of the gland in vivo but that this activity is not required once the structure of the gland is disrupted. An understanding of CMV replication in the salivary gland and how M33 modulates salivary gland function to promote viral replication will provide insights into mechanisms that cytomegaloviruses use to promote persistence and access to saliva, a main bodily fluid by which horizontal viral transmission occurs.

Human cytomegalovirus (HCMV), a beta-herpesvirus, is an important opportunistic pathogen that primarily affects individuals with compromised or immature immune systems. It is of great significance in AIDS patients where it can cause serious morbidity through retinitis-associated blindness, and other complications, such as pneumonia and enteritis. In developed nations, it is a leading viral cause of congenital disease, where in-utero infection manifests in mental and behavioral disorders. In order to control infection and HCMV associated disease, new compounds and novel strategies must be developed. Understanding the role viral proteins play during the course of infection will help elucidate the mechanisms of HCMV pathogenesis and provide important information on potential targets for new treatments. However, the strict species specificity of HCMV prevents any studies into the pathogenesis of the virus in an animal host. This limitation can be overcome through the use of murine cytomegalovirus (MCMV). MCMV, like HCMV, is a betaherpesvirus that exhibits similar pathogenesis in mice to HCMV infection in the human host. The genetic structure of MCMV contains significant sequence homology to HCMV AD169 in at least 78 ORFs and can thereby be used as an important tool in elucidating the functions of these ORFs in a complete in vivo system. In our study, we have conducted a comprehensive YTH screen to identify potential interactions between approximately 170 MCMV ORFs. Growth phenotype analysis were also conducted using five different cell lines potentially involved in various aspects of CMV infection. Between these 170 predicted proteins we have identified 94 potential interactions that exhibit varying levels of essentiality depending on the type of cell infected. We aim to understand the nature of the interactions between the viral particle and proteins encoded by the virus in order to elucidate potential mechanisms by which these proteins help to assemble and create new progeny viruses. The interactions that we have identified in this study provide a framework to predict the functions of uncharacterized viral proteins. And understanding the importance of each protein in the context of infection can further help to determine the nature of these unknown viral proteins. Together using information about known viral proteins that interact with these unknown elements, we can develop a better understanding of how all of these components contribute to viral infection which can be used to determine more effective methods to treat or prevent CMV associated diseases.