The long-term objective of this research program is to elucidate the function of human cytomegalovirus (HCMV) genes that regulate the interaction of the virus with its host cell and thereby control the processes of viral replication and pathogenesis. This proposal will study HCMV proteins that are delivered to the cell as constituents of the virion. These proteins have the potential to exert profound effects on the virus-host interaction, because they are present at the very start of the infectious process. Our studies will be performed in fibroblasts, the standard host cell used for HCMV studies in the laboratory, and in epithelial cells, a cell type that is central to HCMV replication and spread in infected people. We will investigate both laboratory strains and clinical isolates of HCMV. Our technical approach will combine genetics, molecular biology and proteomics.
Our specific aims focus on the functional analysis of three virus-coded virion proteins: pUL83, pUS22 and pUL23/pUL24. pUL83 is the most abundant virion constituent. We and others have shown that it inhibits the induction of cellular anti-viral genes at the start of infection, and we have discovered that it interacts with the cellular IFI-16 protein. We propose to investigate the mechanism by which pUL83 blocks the protective cellular response and how its interaction with IFI- 16 contributes to its function. pUS22 has no known effect on HCMV replication in fibroblasts. We now have discovered that it is required for efficient replication in epithelial cells, and we have shown that it binds to the cellular SF2/ASF protein, a multi-functional splicing factor. We will investigate the host range function of pUS22 in epithelial cells, and we will explore the consequences of its interaction with SF2/ASF. Since depletion of SAF2/ASF and HCMV infection are both known to induce genomic instability, we will test the hypothesis that pUS22 blocks SF2/ASF function and thereby induces genomic instability. We have shown that the pUL23 and pUL24 proteins form a complex and that this complex functions after the viral genome reaches the nucleus and before immediate-early mRNAs function. We will use mutant viruses to further delineate the very early point in the replication cycle at which this protein complex functions, we will identify cellular proteins with which the complex interacts, and we will explore the functional consequences of these interactions.
Human cytomegalovirus (HCMV) infections in healthy children and adults are generally asymptomatic, but the virus causes life-threatening disease in immunologically immature or compromised individuals. Congenital HCMV infection is the leading viral cause of birth defects, neonates can suffer serious complications following infection, and HCMV is a major complication in immunosuppressed individuals, with a significant contribution to morbidity and mortality in allogeneic transplant recipients and AIDS patients. This proposal is designed to enhance our basic understanding of HCMV replication strategies and how the virus antagonizes our anti-viral protective measures.
|Sharon-Friling, Ronit; Shenk, Thomas (2014) Human cytomegalovirus pUL37x1-induced calcium flux activates PKC?, inducing altered cell shape and accumulation of cytoplasmic vesicles. Proc Natl Acad Sci U S A 111:E1140-8|
|Koyuncu, Emre; Purdy, John G; Rabinowitz, Joshua D et al. (2013) Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny. PLoS Pathog 9:e1003333|
|Terry, Laura J; Vastag, Livia; Rabinowitz, Joshua D et al. (2012) Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection. Proc Natl Acad Sci U S A 109:3071-6|
|Liu, Sean T H; Sharon-Friling, Ronit; Ivanova, Pavlina et al. (2011) Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress. Proc Natl Acad Sci U S A 108:12869-74|
|O'Connor, Christine M; Shenk, Thomas (2011) Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient spread by the extracellular route but not for direct cell-to-cell spread. J Virol 85:3700-7|
|Cristea, Ileana M; Moorman, Nathaniel J; Terhune, Scott S et al. (2010) Human cytomegalovirus pUL83 stimulates activity of the viral immediate-early promoter through its interaction with the cellular IFI16 protein. J Virol 84:7803-14|
|Moorman, Nathaniel J; Sharon-Friling, Ronit; Shenk, Thomas et al. (2010) A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation. Mol Cell Proteomics 9:851-60|
|Lilja, Anders E; Shenk, Thomas (2008) Efficient replication of rhesus cytomegalovirus variants in multiple rhesus and human cell types. Proc Natl Acad Sci U S A 105:19950-5|
|Munger, Joshua; Bennett, Bryson D; Parikh, Anuraag et al. (2008) Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol 26:1179-86|
|Lilja, Anders E; Chang, W L William; Barry, Peter A et al. (2008) Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor. J Virol 82:2170-81|
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