Phillips, Stacia L.2012-08-222012-08-222012-06https://hdl.handle.net/11299/131927University of Minnesota Ph.D. dissertation. June 2012. Major: Microbiology, Immunology and Cancer Biology. Advisor: Wade A. Bresnahan, Ph.D. 1 computer file (PDF); ix, 144 pages.Human cytomegalovirus (HCMV) is the largest and most complex of the human herpesviruses, both structurally and with respect to the coding capacity of the viral genome. Proteomic analysis of purified virions suggests that HCMV particles may be composed of as many as 71 virally encoded proteins. Despite intense investigation, the mechanisms by which HCMV particles are assembled in the cytoplasm of infected cells remain poorly understood. In an attempt to gain novel insight into the processes involved in virion assembly, we carried out a yeast two-hybrid screen designed to identify binary interactions among HCMV structural proteins. One interaction of particular interest that was identified in our screen was that between the herpesvirus-conserved tegument proteins UL94 and UL99. UL99 is essential for HCMV replication and functions at the stage of secondary envelopment in the cytoplasm. The function of UL94 is unknown. We hypothesize that the interaction between UL94 and UL99 is essential for HCMV replication. To investigate this hypothesis, we first sought to elucidate the function of UL94. In the absence of UL94, early events such as viral gene expression and DNA replication proceeded at wild type levels. However, we observed a dramatic defect in the localization of UL99 to the viral assembly complex in the cytoplasm of cells infected with the UL94-null mutant. In addition, ultrastructural analysis of cytoplasmic virus particles showed that in cells infected with the UL94-null mutant, there was a complete absence of enveloped virions, demonstrating that UL94, like UL99, is required for secondary envelopment. Finally, we mapped the domains of UL94 and UL99 that are required for their interaction. We then incorporated mutations that abolish the interaction into the viral genome. We showed that when the interaction between UL94 and UL99 was abolished during HCMV infection, both proteins exhibited aberrant localization and the production of infectious virus progeny was completely blocked. Taken together, our results suggest that UL94 functions at least in part to direct the proper localization of UL99 to the assembly complex through their interaction and that this event is essential for HCMV replication.en-USAssemblyEnvelopmentHCMVTegumentVirionMicrobiology, Immunology and Cancer BiologyThesis or Dissertation