Human Cytomegalovirus Use and Manipulation of Host Phospholipids Item Type text; Electronic Thesis Authors Harwood, Samuel John Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 22:10:36 Link to Item http://hdl.handle.net/10150/632563 HUMAN CYTOMEGALOVIRUS USE AND MANIPULATION OF HOST PHOSPHOLIPIDS by Samuel Harwood ____________________________ Copyright © Samuel Harwood 2019 A Thesis Submitted to the Faculty of the DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 2019 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Master's Committee, we certify that we have read the thesis prepared by Samuel Harwood, titled Human Cytomegalovirus Use and Man.!.e.ulationof Host Phos holipids, and recommend that it be accepted as fulfilling the thesis requirement for the Master's Degree. Z'i Date +/ I Z.OI � . 4/ 1 / 7 Date: 2 r ( Date: � /L<I' IC, Final approval and acceptance of this thesis is contingent upon the candidate's submission of the final copies of the thesis to the Graduate College. I hereby certify that I have read this thesis prepared under my direction and recommend that it be accepted as fulfilling the Master's requirement. 4/ 1 '��v Date: 2 I 2ol � Dr. John Purdy Assistant Professor Department of lmmunobiology 3 Acknowledgments I would like to thank Dr. John Purdy, Lisa Wise, Elizabeth Dahlmann, and Yuecheng Xi for their mentorship and assistance with my research. I would also like to thank Dr. Joyce Schroeder and Dr. Daniela Zarnescu for serving on my thesis committee. 4 Table of Contents List of Figures and Tables……………………………………………………………………………………………………………………….5 Abstract………………………………………………………………………………………………………………………………………………….6 Introduction……………………………………………………………………………………………………………………………………………7 Materials and Methods………………………………………………………………………………………………………………………….20 Results………………………………………………………………………………………………………………………………………………..…27 Discussion……………………………………………………………………………………………………………………………………………..49 References…………………………………………………………………………………………………………………………………………….57 5 List of Figures and Tables Table 1…………………………………………………………………………………………………………………………………………………..13 Figure 1………………………………………………………………………………………………………………………………………………….16 Figure 2………………………………………………………………………………………………………………………………………………….31 Figure 3………………………………………………………………………………………………………………………………………………….34 Figure 4………………………………………………………………………………………………………………………………………………….35 Figure 5………………………………………………………………………………………………………………………………………………….39 Figure 6……………………………………………………………………………………………………………………………………………….…40 Figure 7………………………………………………………………………………………………………………………………………………….44 Figure 8………………………………………………………………………………………………………………………………………………….46 Figure 9………………………………………………………………………………………………………………………………………………….48 6 Abstract Human cytomegalovirus (HCMV) is a widely-spread β-herpesvirus that causes a congenital infection that results in devastating disabilities in newborns. Infection also causes life-threatening disease in people with compromised immune systems. HCMV requires viral remodeling of host cell metabolism to obtain metabolites and lipids to support replication and contains an envelope made of lipids ‘stolen’ from the host. Envelopment is a crucial step in the HCMV life cycle and requires numerous host cell lipids, most prominently phospholipids. I hypothesized that HCMV upregulates the production of cellular phospholipids and uses host cell lipid transport proteins to transport those phospholipids to the viral envelope. I used tandem LC-MS/MS to quantitatively determine HCMV manipulation of the abundance of phospholipids. I found that HCMV significantly upregulates phosphatidylcholine (PC) lipids and identified a required viral protein: pUL37x1. PC lipids are transferred between membranes by phosphatidylcholine transfer protein (PC-TP). I found that HCMV upregulates PC-TP expression during infection, suggesting that it is important to infection. To determine the role of PC-TP in virus replication, I used a small molecule inhibitor and a CRISPR/Cas9 knockout of PC-TP. My preliminary data indicates that the loss of PC-TP activity reduces HCMV infection late in the viral replication cycle, consistent with my hypothesis. Additionally, I analyzed how the loss of PC-TP affects HCMV remodeling of host lipid metabolism. Overall, my findings demonstrate that HCMV upregulates cellular phospholipids and that PC-TP may play a role in transporting those lipids in viral replication 7 Introduction Human cytomegalovirus (HCMV) is a globally ubiquitous β-herpesvirus infecting over 60% of the world population [1] with prevalence varying based on location and socioeconomic status (i.e. <90% of preschool children are infected in the developing world compared to 20% in developed countries) [2]. Generally, HCMV infection is asymptomatic for healthy and immunocompetent children, adults, and newborns. However, HCMV is the most common congenital viral infection and can result in deafness, blindness, liver abnormalities, and severe learning disabilities in children. In the United States, HCMV is the leading cause of birth defects [3]. Infection has also been associated with glioblastoma [4, 5], cardiovascular disease [6], and the deterioration of the immune system [7]. Infection can lead to prolonged drug treatment, hospitalization and, in some cases, death. HCMV is also a life-threatening opportunistic infection in immunocompromised patients, including HIV/AIDS patients and stem- cell/solid-organ transplant recipients [1, 8]. HCMV is typically transmitted by the exchange of bodily fluids [9], including sexual activity [10]. HCMV infection can be limited by widespread sanitation (e.g. washing hands, not sharing food and drink-ware), but this practice is impractical on a large scale [11]. There is no cure or vaccine for HCMV. Like all herpesviruses, once a person is infected, they will harbor the HCMV for life since the immune system fails to clear the virus [12]. HCMV has a double-stranded DNA genome surrounded by a capsid, a proteinaceous layer called the tegument, and an envelope composed of host derived lipids and viral proteins. HCMV encodes at least 150 known proteins which are classified as either immediate early, early, or late. However, the virus may encode an additional 400 genes [13]. With a genome length of approximately 236 kbp, HCMV has the largest genome of any herpesvirus. To suppress HCMV infection, patients are typically give acyclovir-related drugs (nucleoside analogues) [14] that include drugs such as ganciclovir, valganciclovir, foscarnet, and cidofovir [15]. These drugs are all related by their competitive inhibition of the viral DNA polymerase [16, 17] and attempt to prevent viral gene synthesis. Although these drugs have improved 8 outcomes across the globe, particularly in immunocompromised hosts, there are several drawbacks to these therapies, particularly that of the associated toxicity of the drugs [15] (leading to the onset of conditions such as cytopenia [14]) as well as the frequent development of anti-viral resistance. The issues of toxicity and viral resistance are connected. Because of the toxicity of the drugs, only limited doses can be administered to patients. As a result, viral replication is incompletely suppressed, resulting in a higher chance of resistant progeny [16]. Interestingly, one of the most commonly prescribed drug combinations, ganciclovir and valganciclovir (GCV), requires a phosphorylation step catalyzed by the viral kinase UL97 [18], offering two points of resistance: mutation in UL97 (accounting for 90% of GCV resistance cases) [19] or the viral DNA polymerase. This leads GCV therapy to having more cases of anti- viral resistance than any other HCMV therapy. These drawbacks have created a need for novel therapies that are less prone to viral resistance as well as less toxic to the host [15]. HCMV modulation of host cell metabolism is one possible therapeutic target. HCMV manipulation of host cell metabolism likely plays a role HCMV virion assembly, particularly that of envelopment. Assembly starts in the nucleus and continues in a virally generated perinuclear organelle called the viral assembly compartment that contains various host and virus proteins and host membranes [1]. The formation of this compartment has been shown to not only increase the expression of organelle markers but also relocate them to the perinuclear site of virion assembly [20]. HCMV undergoes two distinct and independent envelopment phases: primary envelopment that occurs at the inner nuclear membrane of as the viral capsid exits the nucleus (this envelope is only temporary as it is lost at the outer nuclear membrane), and a final envelopment that likely happens in the assembly complex. During final envelopment, lipids made by host metabolic pathways are used to form an infectious HCMV virion [1]. HCMV upregulates the expression of host proteins, including fatty acid elongase 7 enzyme (ELOVL7), to meet the lipid requirements of infection [21]. ELOVL7 produces saturated very long chain 9 fatty acids that are found in the viral envelope [21] and are required for infection [22]. HCMV has also been shown to upregulate PKR-like endoplasmic