In Vivo Labelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication.

Tetsuro Komatsu,Irene Carlon-Andres,Floriane Lagadec,Ralph H Kehlenbach,Wenli Zhang,Fabienne Rayne,Kerstin Bystricky,Franck Gallardo,Jean-Michel Lagarde,Renaud Morin,Jessica Ragues,Charlotte Quentin-Froignant,Harald Wodrich,Anja Ehrhardt,Lawrence Banks

doi:10.1128/jvi.00795-18

Abstract

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 in vivo 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 in vivo 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 in vivo 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.IMPORTANCE 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 in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo 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.

Highlights

Adenoviruses are DNA viruses with a lytic infection cycle
Replication of AdV DNA occurs through a single-stranded intermediate and coincides with the formation of replication centers (RC), which are morphologically identifiable by the single-stranded DNA binding protein (DBP) or cellular marker proteins, such as the ubiquitin-specific protease USP7 [28,29,30]
Using specific markers for early and late RC, we further showed that AdV genome replication occurs in two kinetically distinct phases associated with a switch in RC morphology

Summary

Introduction

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. Attempts to genetically label AdV genomes for in vivo imaging used multiple copies of the tet operator, replacing the E1 region and E1-complementing cells expressing green fluorescent protein (GFP)-tagged tet repressor This system allowed labeling of capsidassociated genomes from incoming particles in living cells in real time but failed to detect genomes at later stages of infection, e.g., upon or after nuclear import [34]. We exploited the TAF-I␤–protein VII association and showed that cell lines expressing GFP-tagged TAF-I␤ form spots in the nucleus, depicting single incoming genomes in living cells [41] Using this first functional in vivo imaging system for individual intranuclear AdV chromatin complexes, we showed that AdV avoids recognition by most known nuclear DNA sensors and prevents transcriptional silencing [39, 44, 45]. The system requires genomebound protein VII, and its removal, e.g., upon replication, limits in vivo observations to the early infection phase

Methods

Results

Conclusion