Viruses have evolved elegant strategies to manipulate host cell machinery and rewire core cellular pathways to facilitate productive infection, including enhancing metabolic output and maintaining cell viability. To accomplish this, viruses exert an extensive network of dynamic molecular interactions with cellular organelles. As the functions of organelles are intimately associated with the regulation of their composition, shape, and localization, the control of organelle structure-function relationships is at the core of clarifying the outcome of an infection. While many examples of virus-induced organelle remodeling have been described, very little is understood about how organelle structures engender specific functions. Our lab has characterized a previously unrecognized aspect of viral infection, which is that human viruses globally control organelle remodeling by dramatically rewiring inter- and intra-organelle membrane contact sites (MCS). Using a hybrid quantitative proteomics and super resolution microscopy approach, we demonstrated exquisite reorganization in MCS networks engaged by a broad range of human viruses, including both ancient (herpesviruses) and rapidly adapting (influenza and beta- coronavirus) viruses. We further discovered that infection with the ubiquitous herpesvirus human cytomegalovirus (HCMV) triggers a new specialized MCS structure, mitochondria-ER encapsulations that we termed MENC. We determined that HCMV infection drives predominantly fission at the mitochondrial periphery, and that the fragmented mitochondria enter MENCs and retain their bioenergetic activity. How the infection induces MENC formation and the function of this newly reported structure remain unknown. We propose that MENCs provide a unifying explanation for the longstanding paradox of how certain viruses such as HCMV increase mitochondrial bioenergetic output, despite inducing mitochondrial fragmentation. Our central hypothesis is that HCMV remodels inter- and intra-organelle connections, generating MENCs, which act to protect and stabilize the bioenergetic capacity of fragmented mitochondria. Using a multidisciplinary approach that combines molecular virology with cutting-edge approaches in quantitative proteomics, live super resolution microscopy, ultrastructural electron microscopy, metabolomics, and lipidomics, in Aim 1, we will define the mechanisms underlying the formation and function of MENCs during HCMV infection. In Aim 2, we will establish what signaling cues from HCMV-induced three-way contacts among the ER, mitochondria, and lysosome stimulate peripheral mitochondria fission and elevate bioenergetic respiration. In Aim 3, we will characterize the viral factors that coordinate ER-mitochondria MCS rewiring. Collectively, our study will link newly discovered aspects of virus-orchestrated MCS networking to new two-way and three-way organelle structure-function relationships that underlie fundamental cellular mechanisms, including mitochondri...