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<jats:title>ABSTRACT</jats:title><jats:p>Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile<jats:italic>in vivo</jats:italic>imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with<jats:italic>in vivo</jats:italic>genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes<jats:italic>in vivo</jats:italic>. The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase.</jats:p><jats:p><jats:bold>IMPORTANCE</jats:bold>Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an<jats:italic>in vivo</jats:italic>DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the<jats:italic>in vivo</jats:italic>visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.</jats:p>

Original publication




Journal article


Journal of Virology


American Society for Microbiology

Publication Date