The HCMV Assembly Compartment Is a Dynamic Golgi-Derived MTOC that Controls Nuclear Rotation and Virus Spread

Human cytomegalovirus (HCMV), a leading cause of congenital birth defects, forms an unusual cytoplasmic virion maturation site termed the “assembly compartment” (AC). Here, we show that the AC also acts as a microtubule-organizing center (MTOC) wherein centrosome activity is suppressed and Golgi-based microtubule (MT) nucleation is enhanced. This involved viral manipulation of discrete functions of MT plus-end-binding (EB) proteins. In particular, EB3, but not EB1 or EB2, was recruited to the AC and was required to nucleate MTs that were rapidly acetylated. EB3-regulated acetylated MTs were necessary for nuclear rotation prior to cell migration, maintenance of AC structure, and optimal virus replication. Independently, a myristoylated peptide that blocked EB3-mediated enrichment of MT regulatory proteins at Golgi regions of the AC also suppressed acetylated MT formation, nuclear rotation, and infection. Thus, HCMV offers new insights into the regulation and functions of Golgi-derived MTs and the therapeutic potential of targeting EB3. •HCMV assembly compartments (ACs) are dynamic Golgi-derived MTOCs•Viral IE2 protein and CDK regulation increase host EB protein levels for MT control•AC structure and nuclear rotation are controlled by EB3-regulated acetylated MTs•A cell-permeable peptide targeting EB3 suppresses HCMV replication Live-cell imaging reveals the dynamic nature of the human cytomegalovirus (HCMV) assembly compartment (AC), which acts as a Golgi-derived microtubule-organizing center. Specific recruitment of microtubule plus-end binding EB3 facilitates acetylated MT formation at the AC and nuclear rotation prior to cell migration. EB3 targeting with myristoylated peptides suppresses infection.