Study of the interferon-oxysterol antiviral response and 3-Hydroxy-3-Methylglutaryl-CoA Reductase
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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.