Abstract
Biomolecular assemblies are fundamental to life and viral disease. The spatiotemporal coordination of viral replication and assembly is largely unknown. Here, we developed a dual-color click chemistry procedure for imaging adenovirus DNA (vDNA) replication in the cell nucleus. Late- but not early-replicated vDNA was packaged into virions. Early-replicated vDNA segregated from the viral replication compartment (VRC). Single object tracking, superresolution microscopy, fluorescence recovery after photobleaching, and correlative light-electron microscopy revealed a stepwise assembly program involving vDNA and capsid intermediates. Depending on replication and the scaffolding protein 52K, late-replicated vDNA with rapidly exchanging green fluorescent protein-tagged capsid linchpin protein V and incomplete virions emerged from the VRC periphery. These nanogel-like puncta exhibited restricted movements and were located with the capsid proteins hexon, VI, and virions in the nuclear periphery, suggestive of sites for virion formation. Our findings identify VRC dynamics and assembly intermediates, essential for stepwise productive adenovirus morphogenesis.