FOLLOWING THE FATE OF HCMV GENE PRODUCTS IN HOST CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytomegalovirus is widely spread throughout all geographic locations and socioeconomic group and infects 85% of adults in United States by 40 years of age. Our aim is to study the interactions of human cytomegalovirus with cellular proteins, and gain insights into cellular pathways modulated by herpesviruses. This study was initiated one and a half years ago in collaboration with the laboratory of Tom Shenk at Princeton University. Since then, we have isolated a variety of Protein A- or GFP-tagged HCMV viral proteins identifying their host and viral interacting partners at various stages of infections. During this last year, we have studied the interacting partners of several viral proteins, including UL83, UL121, UL38, UL99, and US22. Each of these studies highlighted new ways that HCMV utilizes to manipulate and take over the cell. Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress. A manuscript describing this work has been published: Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T. Cell Host Microbe. 2008 3(4):253-62.