A lipidomic and proteomic analysis of human cytomegalovirus

Human cytomegalovirus has developed a vast arsenal of proteins to manipulate existing pathways within target host cells. This thesis takes two approaches towards studying the interplay between human cytomegalovirus and its host.The first approach focuses on how the virus influences global changes on the cell. Previous studies had suggested that human cytomegalovirus may upregulate fatty acid production for efficient viral replication. Working in collaboration with the Rabinowitz and Brown laboratories, I used mass spectrometry to quantify the presence of 146 glycerolphospholipid species in mock-infected and virus-infected whole cell lysates over a 4-day timecourse. The majority of glycerolphospholipid species did not change in infected samples compared to mock samples. Using similar techniques, I measured the lipid composition of purified human cytomegalovirus virions. The distribution of the glycerolphospholipid headgroup classes was noticeably different in the virions versus the cell lysates. When compared to a variety of previously published lipid datasets, the virion datasets showed the greatest similarities to synaptic vesicle lipidomes isolated from rat brains. This prompted an inquiry of whether human cytomegalovirus may use export machineries in fibroblasts that are analogous to the neurotransmitter export pathways found in neurons. Knockdown studies with targeted siRNAs against SNAP23 showed that efficient viral replication depended on this vesicular machinery. Furthermore, co-immunoprecipitation established an interaction between SNAP23 and the essential tegument protein, pp28. Additional studies will be needed to characterize the functional consequence of this interaction.The second approach looks at how a single viral protein can interact with numerous cellular pathways. The immediate-early viral gene product, pUL37x1, is best known for its inhibitory role against apoptosis through its interaction with the pro-apoptotic protein, Bax. However, many additional uncharacterized functions have been ascribed to pUL37x1, such as its involvement in host cytoskeletal rearrangement and intracellular calcium regulation. Working in collaboration with the Cristea laboratory, I used mass spectrometry combined with GalK recombineering to identify a proteomic list of candidate interactions to pUL37x1. Of the identified candidates, a novel interaction with VDAC protein may help characterize the role of pUL37x1 in intracellular calcium regulation.