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.
|Purdy, John G; Shenk, Thomas; Rabinowitz, Joshua D (2015) Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication. Cell Rep 10:1375-85|
|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|
|Mathias, Rommel A; Greco, Todd M; Oberstein, Adam et al. (2014) Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity. Cell 159:1615-25|
|Grady, Sarah L; Purdy, John G; Rabinowitz, Joshua D et al. (2013) Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection. Proc Natl Acad Sci U S A 110:E5006-15|
|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|
|Lee, Song Hee; Kalejta, Robert F; Kerry, Julie et al. (2012) BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection. Proc Natl Acad Sci U S A 109:9575-80|
|Gudleski-O'Regan, Nicole; Greco, Todd M; Cristea, Ileana M et al. (2012) Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity. Cell Host Microbe 12:86-96|
|Grady, Sarah L; Hwang, Jesse; Vastag, Livia et al. (2012) Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase. J Virol 86:8259-68|
|O'Connor, Christine M; Shenk, Thomas (2012) Human cytomegalovirus pUL78 G protein-coupled receptor homologue is required for timely cell entry in epithelial cells but not fibroblasts. J Virol 86:11425-33|
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