A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

Komatsu, Tetsuro; Dacheux, Denis; Kreppel, Florian; Nagata, Kyosuke; Wodrich, Harald

doi:10.1371/journal.pone.0137102

Cited by 31 publications

(64 citation statements)

References 27 publications

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“…The expression vectors for enhanced green fluorescent protein (EGFP)-TAF-I␤, HA-DBP, and HA-DBP⌬C (pEGFP-C1-TAF-I␤, pCHA-puro-DBP, and pCHA-puro-DBP⌬C) are described elsewhere (29,35). For the construction of the Daxx expression vector, the cDNA fragment for Daxx was amplified by PCR, digested with BamHI and EcoRI, and inserted into the pCHA-puro vector (35) (pCHA-puro-Daxx) and subsequently combined with a N-terminal insertion of the FLAGmCherry tag.…”

Section: Methodsmentioning

confidence: 99%

“…Indirect immunofluorescence (IF) and live-cell imaging analyses were carried out as described previously (29). IF samples were analyzed by a Leica SP5 confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

“…To examine whether incoming Ad genome complexes localize at PML-NBs, we performed IF analyses using antibodies against PML-NB components and protein VII, a marker for viral genome complexes (29) (Fig. 1).…”

Section: Incoming Ad Genome Complexes Do Not Localize At Pml-nbs Durimentioning

confidence: 99%

“…1), it remained possible that incoming Ad genome complexes only transiently localize at PML-NBs and/or recruit its components-for instance, upon nuclear import, as reported for HSV-1 (13). To test this possibility, we next tested the association of incoming Ad genome complexes with PML-NBs in living cells using our recently developed live-cell imaging system (29). The analysis is based on the use of fluorescently labeled TAF-I, a cellular chromatin protein binding to protein VII upon Ad infection (41).…”

Section: Incoming Ad Genome Complexes Do Not Localize At Pml-nbs Durimentioning

confidence: 99%

“…Protein V appears to be lost before the nuclear import of the genomes (28), while the fate of polypeptide X/Mu is unclear. Thus, for the first hours after nuclear import, protein VII marks viral genome complexes in cells (29). An early report suggested that incoming Ad genomes localize at PML-NBs (5).…”

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An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

Komatsu

Nagata

Wodrich

2016

J Virol

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Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. IMPORTANCEThe immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes.

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Section: Methodsmentioning

confidence: 99%

“…Indirect immunofluorescence (IF) and live-cell imaging analyses were carried out as described previously (29). IF samples were analyzed by a Leica SP5 confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

Section: Incoming Ad Genome Complexes Do Not Localize At Pml-nbs Durimentioning

confidence: 99%

Section: Incoming Ad Genome Complexes Do Not Localize At Pml-nbs Durimentioning

confidence: 99%

mentioning

confidence: 99%

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An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

Komatsu

Nagata

Wodrich

2016

J Virol

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Formation of adenovirus DNA replication compartments

Hidalgo

González

2019

FEBS Letters

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Adenoviruses induce an extensive reorganization of the host cell nucleus during replication. Such a process results in the assembly of viral and cellular macromolecules into nuclear structures called adenovirus replication compartments (AdRCs), which function as platforms for viral DNA replication and gene expression. AdRCs co-opt host proteins and cellular pathways that restrict viral replication, suggesting that the mechanisms that control AdRC formation and function are essential for viral replication and lay at the basis of virus-host interactions. Here, we review the hallmarks of AdRCs and recent progress in our understanding of the formation, composition, and function of AdRCs. Furthermore, we discuss how AdRCs facilitate the interplay between viral and cellular machineries and hijack cellular functions to promote viral genome replication and expression.

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Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

Cited by 31 publications

References 27 publications

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

Formation of adenovirus DNA replication compartments

Imaging the adenovirus infection cycle

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