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

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.

Keywords: DNA tagging; adenovirus; chromatin association; in vivo labelling; incoming genome; live cell imaging; nuclear transport; viral replication.

FIG 1

Detection of incoming AdV genomes through direct DNA labeling. (A) Schematic overview of ANCHOR3 encoding AdV vector with E1/E3 deleted. The OR3-GFP expression cassette (light gray) is upstream of the Anch sequence consisting of 10 nucleation seeds for binding and oligomerization of the OR3-GFP protein (green spheres). (B) Coomassie gel analysis of purified virus particles without (lane 1) or with (lane 2) inserted ANCHOR3 sequence. Major capsid proteins are indicated on the right. (C) U2OS (top row) or H1299 (bottom row) epithelial cells were transfected with OR3-GFP expression plasmid and either mock infected (a and d) or infected with Ad5-ΔE1ΔE3-ANCHOR3-GFP (b,c,e, and f). At 3 hpi, cells were imaged by spinning-disk microscopy, and individual frames are shown. OR3-GFP spots representing viral genomes are indicated by arrows (see Movie S1A to C in the supplemental material). (D) Stable mCherry-TAF-Iβ-expressing U2OS cells were transfected with OR3-GFP expression plasmid and either mock infected (top row), infected with Ad5-ΔE1ΔE3-GFP (middle row), or infected with Ad5-ΔE1ΔE3-ANCHOR3-GFP (bottom row). At 3 hpi, the cells were imaged by spinning-disk microscopy, and individual frames are shown. An overlay of the GFP signal (left column) and the mCherry signal (middle column) is shown in the right column. Individual genomes are indicated by white arrows and their hypothetical positions by gray arrows. (E) As in panel D using OR3-GFP-transfected U2OS cells. Cells were fixed at 3 hpi and stained with anti-TAF-Iβ antibodies. The GFP signal (green) and the TAF-Iβ signal (red) are shown, and individual genomes and their hypothetical positions are indicated by white and gray arrows, respectively. Scale bars, 10 μm.

FIG 2

Dynamic analysis of incoming intranuclear genomes. (A) At 24 hpi, intranuclear OR3-GFP- labeled genomes were imaged by time-lapse confocal microscopy at 1 frame/3 s. A single frame is depicted on the left and a projection over 3 min depicting absence of genome mobility on the right. (B) FRAP analysis of the same nucleus as in panel A showing a single genome before (left) and after (right) bleaching. Individual frames on the bottom are from the boxed region showing OR3-GFP fluorescence recovery on the bleached genome. (A and B) Scale bars, 5 μm. (C) FRAP recovery curves of individual genomes. The green arrow indicates the time of photobleaching application, and the x axis indicates time postbleaching. The red line indicates the best fit of the recovery curve with the 95% CI (dotted blue line; n = 48). The half-recovery time is indicated by the black vertical line (best fit) or the gray box (95% CI). (D) OR3-GFP-expressing cells were infected with Ad5-ΔE1ΔE3-ANCHOR3-GFP and imaged at 1 frame/8 min (see Movie S2 in the supplemental material). Time-stamped individual frames are shown as raw images (left) or following deconvolution (right), depicting breakdown of the permeability barrier of the nuclear envelope (large arrows) and intranuclear genomes (small arrows).

FIG 3

Chromatin anchoring of incoming AdV genomes. (A) Cells were infected with Alexa 488-labeled virus particles (AdV-488; green signal) and fixed at 2 hpi. Viral genomes were stained with protein VII-specific antibodies (VII; red signal), cellular chromatin was stained with DAPI (4′,6-diamidino-2-phenylindole) (gray signal), and the nuclear envelope was stained with lamin A/C-specific antibodies (LAC; cyan signal) as indicated. The top row depicts a cell in interphase, and the middle and bottom rows a cell in mitosis. Scale bars, 5 μm. The white and black arrows indicate chromatin-associated genomes. The dashed arrow indicates a genome still associated with capsid at the nuclear envelope. Note that the images are Z-stack projections. (B) Cells expressing fluorescent histone H2B (tdiRFP; red signal) and OR3-GFP (green signal) were imaged at 3 hpi using live-cell imaging. The large images show mitotic cells using the combined signals; the small images below show separate channels for viral (left; green) and cellular (right; red) chromatin. The green arrows indicate chromatin-associated viral genomes. (C) As in panel B, showing several still images of a live-cell imaging sequence of separating chromosomes with associated viral genomes with ∼1 min between frames (see Movie S3 in the supplemental material).

FIG 4

ANCHOR3-GFP in AdV genome replication in fixed cells. Cells were mock infected or infected with Ad5-ΔE1ΔE3-ANCHOR3-GFP in the absence (top [long] row) or presence (middle and bottom [long] rows) of replicative Ad5-ΔE, as indicated on the top and left. At 24 hpi, cells displaying GFP fluorescence (green signal) were fixed and stained with DBP-specific antibodies (red signal). The long middle row shows cells with early RC and the long bottom row cells with late RC as defined previously (32). The two short rows at the bottom show enlargements of the boxed region in the last column depicting replicated DNA in early (I) and late (II) RC, respectively.

FIG 5

ANCHOR3-GFP in AdV genome replication in living cells. (A) Cells stably expressing mCherry-USP7 display RC upon infection. Representative fields of view are shown at 3-h intervals for high MOI and for lower MOI at 1:10 dilution. The graph shows the absolute number of cells starting replication from 10 independent fields of view. Note that at high MOI, all the cells replicate by ∼15 hpi. (B) Stable mCherry-USP7-expressing cells were infected with an excess of Ad5-ΔE1ΔE3-ANCHOR3-GFP in the presence of replicative Ad5-ΔE3. A representative cell is shown, using individual frames from live-cell imaging at 3-h intervals (12 to 23.5 hpi), as indicated. The spatiotemporal organization of RC (top row; USP7 signal; magenta) and replicated genomes (middle row; OR3-GFP signal; green) and the spatial relationship of both signals (bottom row) are shown (see Movie S4 in the supplemental material). The boxed areas display formation of ViPR bodies, indicated by white arrows in the last panel (see the text for details). Scale bars, 10 μm.

FIG 6

Biphasic AdV genome replication. (A) Stably mCherry-USP7-expressing cells were infected with a mixture of replicative Ad5-ΔE3 with an excess of Ad5-ΔE1ΔE3-ANCHOR3-GFP vector. The images are individual frames showing the development of the OR3-GFP signal during AdV replication. The depicted time points (a to f) are as indicated in panel B. (B) Number of detected genomes measured in individual OR3-GFP signals using a representative cell (shown in panel A) over time. The x axis depicts time postinfection. The dotted vertical lines and letters indicate the time points of the respective frame shown in panel A. (C) Mean genome copy numbers estimated from the OR3-GFP signals from 10 cells. The signals were normalized and set to t₀ at the onset of replication. The error bars indicate standard errors of the mean (SEM). (D) Numbers of genome copies present in the OR3-GFP signals during early/slow (left) and late/fast (right) replication phases. The absolute number of genomes and timing were normalized to the same starting point. Slow and fast replication phases for individual cells are depicted on both graphs in the same colors (black for high MOI and green for low MOI, as indicated in the legend). (E) Replication rates from individual cells in panel D were calculated by linear least-squares regression individually for low and high MOI, as well as for slow and fast replication phases. The values are derived from cells infected at high MOI (black) or low MOI (green). The indicated values correspond to the means and standard deviations (SD) for high replication rates and low replication rates (n = 10 cells).

FIG 7

RC dynamics during AdV replication. (A) Stably mCherry-USP7-expressing cells were infected with a mixture of replicative Ad5-ΔE3 with an excess of Ad5-ΔE1ΔE3-ANCHOR3-GFP vector. The images are individual frames showing the mCherry-USP7 signal indicative of the RC during AdV replication. The depicted time points (a to f) are as indicated in panel B. (B) Quantification of the RC number (blue graph) and RC average size (magenta graph) from a representative cell (shown in panel A) over time. The x axis depicts time postinfection. The dotted vertical lines and letters indicate the time points of the respective frames shown in panel A. (C) Quantification of RC numbers (blue graph) and RC average sizes (magenta graph) from 10 cells. Signals were normalized and set to t₀ at the onset of RC formation. The error bars indicate SEM.

FIG 8

Mybbp1A reorganization during AdV replication. (A) Stably mCherry-Mybbp1A-expressing cells were infected with a mixture of replicative Ad5-ΔE3 with an excess of Ad5-ΔE1ΔE3-ANCHOR3-GFP vector. The images are individual frames showing the mCherry-Mybbp1A signal during AdV replication. The depicted time points (a to f) are as indicated in panel B. (B) Numbers of Mybbp1A foci (blue graph) and average sizes of Mybbp1A foci (magenta graph) from a representative cell (shown in panel A) over time. The x axis depicts time postinfection. The dotted vertical lines and letters indicate the time points of the respective frame shown in panel A. (C) Number sof Mybbp1A foci (blue graph) and average sizes of Mybbp1A foci (magenta graph) from 10 cells. The signals were normalized and set to t₀ at the onset of genome replication. The error bars indicate SEM. (D) As in panel A, but a different cell showing the Mybbp1A signal (left column; magenta), the OR3-GFP signal (middle column; green), and their spatial relationship in the overlay (right column). The rows (a to d) depict time points as indicated in panel E. (E) Numbers of Mybbp1A foci (blue graph), their average sizes (magenta graph, normalized to detected genomes), and the numbers of detected genomes measured in individual OR3-GFP signal (red graph) from a representative cell (shown in panel D) over time. The x axis depicts time postinfection. The dotted vertical lines and letters indicate the time points of the respective frames shown in panel D. (See Movie S5 in the supplemental material.) (F) Numbers of Mybbp1A foci (blue graph), their average sizes (magenta graph, normalized to detected genomes), and the numbers of detected genomes measured in individual OR3-GFP signals (red graph) as in panel E from a cell in early second replication phase. The x axis depicts time postinfection, and the dotted vertical lines indicate the respective frames shown above the graph at 15 hpi, 18 hpi and 23 hpi. Note the concentration of the signal into ViPR bodies late in infection (arrows). Scale bars, 10 μm.

FIG 9

Association of core proteins with ANCHOR3-GFP-tagged genomes. Cells were infected with a mixture of replicative Ad5-ΔE3 with an excess of Ad5-ΔE1ΔE3-ANCHOR3-GFP vector and fixed at 24 hpi. The cells show replicated genomes (green signal) in ViPR bodies surrounded by USP7 detected with specific antibodies (red signal) and costained with antibodies (cyan signal) against AdV particles (top row), protein V (second row), protein VII (third row), pTP (fourth row), and E4orf6 (bottom row), as indicated on the top and left. Scale bars, 5 μm.