Productive replication of Epstein-Barr virus (EBV) during the lytic cycle occurs in discrete sites within nuclei, termed replication compartments. We previously proposed that replication compartments consist of two subnuclear domains: "ongoing replication foci" and "BMRF1-cores". Viral genome replication takes place in ongoing replication foci, which are enriched with viral replication proteins, such as BALF5 and BALF2. Amplified DNA and BMRF1 protein accumulate in BMRF1-cores, which are surrounded by ongoing replication foci. We here determined the locations of procapsid and genome-packaging proteins of EBV via three-dimensional (3D) surface reconstruction and correlative fluorescence microscopy- electron microscopy (FM-EM). The results revealed that viral factors required for DNA packaging, such as BGLF1, BVRF1, and BFLF1 proteins, are located in the innermost subdomains of the BMRF1-cores. In contrast, capsid structural proteins, such as BBRF1, BORF1, BDLF1, and BVRF2, were found both outside and inside the BMRF1-cores. Based on these observations, we propose a model in which viral procapsids are assembled outside the BMRF1-cores and subsequently migrate therein, where viral DNA encapsidation occurs. To our knowledge, this is the first report describing capsid assembly sites in relation to EBV replication compartments.
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