Human cytomegalovirus (HCMV) is an important human pathogen, causing serious disease in immune compromised individuals and in congenitally infected children. Improved methods are needed for prevention and control of HCMV infections. We recently made the novel observation that the center of the human cytomegalovirus (HCMV) cytoplasmic virion assembly compartment (AC) harbors an assemblage of vesicles that are marked by early endosome antigen 1 (EEA1). This and further work led to development of a new model of AC structure. The AC is created during a dramatic cytoplasmic remodeling process that takes place during the first 2 to 4 days after infection. The remodeling results in reorientation of the cellular exocytic pathway such that the early endocytic compartment ultimately resides at the center of a network of concentrically-arranged Golgi and trans-Golgi vesicles. While initially counterintuitive, the three dimensional AC structure offers a rational pathway of virion maturation and egress. The objective of the proposed work is to identify the HCMV gene product(s) responsible for reorientation of the cellular secretory apparatus in HCMV infected cells. AC development is dependent on prior viral DNA synthesis, indicating that one or more viral Early-Late or Late genes are essential for the process. Thus, in Aim 1 we will systematically ablate expression of viral Early-Late and Late genes that are known to be important for efficient viral replication. Confocal microscopy will be used to assay for the distinctive cytoplasmic rearrangements that are indicators of AC development. In Aim 2, the roles of these proteins will be identified by ectopic expression of candidate genes in uninfected cells, complementation of viruses lacking these genes by ectopic gene expression and by mixed infections (e.g., if two genes are involved, co-infection with viruses singly deleted for the genes in question), and identification of cellular proteins that interact with the viral proteins responsible for AC development. This work will provide (i) deeper understanding of HCMV maturation and egress, (ii) expansion of the array of identified functions for HCMV genes, (iii) identification of cellular participants in this process, and (iv) illumination of mechanisms that control cellular organellogenesis. Because of the importance of the AC to HCMV virion maturation, this work will result in new molecular targets and mechanisms to be exploited by antivirals.