Despite its size and rigidity, the cell nucleus can be moved or reorganized by cytoskeletal filaments under various conditions (for example, during viral infection)(1-11). Moreover, whereas chromatin organizes into non-random domains(12), extensive heterogeneity at the single-cell level(13)means that precisely how and why nuclei reorganize remains an area of intense investigation. Here we describe convolutional neural network-based automated cell classification and analysis pipelines, which revealed the extent to which human cytomegalovirus generates nuclear polarity through a virus-assembled microtubule-organizing centre. Acetylation of tubulin enables microtubules emanating from this centre to rotate the nucleus by engaging cytoplasmically exposed dynein-binding domains in the outer nuclear membrane protein nesprin-2G, which polarizes the inner nuclear membrane protein SUN1. This in turn creates intranuclear polarity in emerin, and thereby controls nuclear actin filaments that spatially segregate viral DNA from inactive histones and host DNA, maximizing virus replication. Our findings demonstrate the extent to which viruses can control the nucleus from the cytoplasm. Human cytomegalovirus rotates the nuclei of infected cells to set up intranuclear polarization and thereby separate viral DNA from inactive histones and associated host DNA.