Proteomic Study of Human Cytomegalovirus Using an Epitope Tag System
Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause significant morbidity in neonates and immunodeficient individuals such as, AIDS patience and organ transplant recipients. With a genome size of 190-230 kilobases in size and an estimated 160 or more protein-encoding genes, HCMV is currently the largest known virus to infect humans. The functions of many of HCMV's genes remain unknown and only by understanding their role in viral infection and replication will we be able to successfully develop therapies to combat the disease.
Using a Bacterial Artificial Chromosome (BAC) containing the genome of HCMV, we constructed over 100 recombinant viruses, each with a single open reading frame (ORF) expressing its protein with an epitope tag at the C terminus end. Using these viruses, we were able to identify subcellular localization for 82 ORFs at 72 hours post infection. Our study found 17 proteins to localize to the nucleus, 51 to a juxtanuclear structure in the cytoplasm, 11 to the cytoplasm, and three to an uncharacterized structure in the cytoplasm.
We then conducted an extensive study of HCMV ORF US20, which exhibited a unique cytoplasmic localization in the previous global localization study. The US20 ORF is a member of the US12 gene family and is present only in cytomegaloviruses strains that infect rhesus monkeys, chimpanzees, and humans, suggesting that the ORF is an important factor for the infection of upper primates. We found the US20 ORF to express a 26-28kDa- seven transmembrane (TM) domain protein during the early/delayed early phase of viral replication, which localized to a cytoplasmic structure during the late phase of replication. This localization was found to be independent of late gene expression. Through immunofluorescence (IFA) studies, we found the US20 protein to be present in early endosomes but not in the ER or TGN, suggesting that the protein is present in membranes of endosomes. However, we do not believe that the protein traffics to the surface of the cell, which we showed by selective membrane permeabilization. The US20 protein was also determined to form homo-dimers, however the function of the dimerization is still unknown. Using viruses expressing truncated forms of US20, which replicated at levels similar to the wild-type, we found that deleting TM5-TM7 significantly reduced US20 protein levels, deleting TM4-TM7 abolished dimerization, and deleting TM2-TM7 destabilized the localization of US20 in the cytoplasm. Through immunoprecipitation and mass spectrometry, we found US20 to associate with valosin containing protein, sodium potassium ATPase, and succinate dehydrogenase. Currently, under the context of infection we have confirmed the interaction of US20 with valosin containing protein (VCP), which has been found to be associated with some neurodegenerative diseases. This interaction presents the hypothesis that US20 may play a role in HCMV congenital infection of the brain.
Lastly, we examined the role of a predicted SUMOylation site located near the C terminus tail of the HCMV processivity factor UL44, which forms a C clamp around the viral DNA. SUMOylation is a post-translation modification that has been found to be involved in various cellular systems, including cell cycle regulation, transcription, cellular localization, degradation, and chromatin organization. In our study, we found that mutating the lysine residue of the conserved SUMOylation motif, ψKxE, at position 410 of UL44 to an alanine did not impair viral DNA synthesis or viral replication. This result indicates that the lysine residue of UL44 is not important for replication of HCMV in fibroblasts.
The results of this dissertation bring new insight into many of HCMV's genes, specifically US20, which prior to this study had not been extensively characterized. The data I present here provides new information about US20 and generates new hypotheses relating to its function. Furthermore, my work here can serve as a foundation for the study of other genes in the US12 gene family.