<i>In Vivo</i>
            Labelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication

Komatsu, Tetsuro; Quentin-Froignant, Charlotte; Carlón-Andrés, Irene; Lagadec, Floriane; Rayne, Fabienne; Ragues, Jessica; Kehlenbach, Ralph H.; Zhang, Wenli; Ehrhardt, Anja; Bystricky, Kerstin; Morin, Renaud; Lagarde, Jean‐Michel; Gallardo, Franck; Wodrich, Harald

doi:10.1128/jvi.00795-18

Cited by 41 publications

(63 citation statements)

References 66 publications

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“…In many cases, the accumulation of these viral proteins throughout the nucleoplasm can be detected prior to the formation of VRCs [16,68,85], suggesting that their formation is concentration dependent, which is another feature of LLPS [87][88][89][90]. HSV-1, CMV, PRV, and HAdV VRCs also coalesce as infection progresses and they grow, fusing together in a liquid-like manner [14,15,61,62,65,69,95,[102][103][104]. However, it was recently demonstrated that HSV-1 VRCs are not disrupted by treatment with 1,6-hexanediol, which is a feature of many other cellular BMCs.…”

Section: Biophysical Processes In Replication Compartment Formationmentioning

confidence: 99%

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

Charman

Weitzman

2020

Viruses

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DNA viruses that replicate in the nucleus encompass a range of ubiquitous and clinically important viruses, from acute pathogens to persistent tumor viruses. These viruses must co-opt nuclear processes for the benefit of the virus, whilst evading host processes that would otherwise attenuate viral replication. Accordingly, DNA viruses induce the formation of membraneless assemblies termed viral replication compartments (VRCs). These compartments facilitate the spatial organization of viral processes and regulate virus-host interactions. Here, we review advances in our understanding of VRCs. We cover their initiation and formation, their function as the sites of viral processes, and aspects of their composition and organization. In doing so, we highlight ongoing and emerging areas of research highly pertinent to our understanding of nuclear-replicating DNA viruses.Viruses 2020, 12, 151 2 of 20 The Initiation and Formation of Replication CompartmentsDNA viruses that replicate in the nucleus exhibit a diverse array of strategies to complete their replication cycle and ultimately produce progeny virions. Despite their many differences, the strategies employed by DNA viruses to initiate productive infection and establish VRCs share many features in common. Indeed, it is likely that all DNA viruses that replicate in the nucleus form VRCs. Following penetration of the host cell and the transport of viral capsids/cores, viral DNA genomes enter the nucleus, where they begin a program of temporally regulated gene expression that initiates productive infection [1,2]. Early gene products include viral proteins required to manipulate the host-cell environment and replicate the viral genome. Viral replication proteins interact with the viral genome and initiate genome replication leading to VRC formation, which is often in concert with certain co-opted host proteins. However, these viruses must overcome several challenges to form VRCs successfully. Firstly, incoming genomes must avoid cellular intrinsic antiviral defenses and homeostatic regulatory pathways such as elements of the DNA damage response (DDR) that respond to the presence of foreign DNA and act to suppress viral gene expression [3][4][5][6][7][8][9][10]. Secondly, VRCs typically form at specific sites within the nucleus, suggesting that viral genomes need to be targeted to these sites in order to initiate VRC formation [6,11,12]. Furthermore, it has been hypothesized that the formation and growth of VRCs may require manipulation of the nuclear environment and the re-organization of host chromatin to overcome the spatial restraints of the nucleus [11][12][13][14][15]. In this section, we review key features of VRC initiation and highlight some major challenges to VRC formation. Interplay between Viral Replication Compartment Formation and Promyelocytic Leukemia Protein Nuclear BodiesA common theme amongst many nuclear-replicating DNA viruses is initiation of VRCs at sites in close proximity to promyelocytic leukemia protein (PML) nuclear bodies (NBs). Since the dis...

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Section: Biophysical Processes In Replication Compartment Formationmentioning

confidence: 99%

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

Charman

Weitzman

2020

Viruses

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“…To overcome this drawback, we set up a new system that allowed direct tracking of viral retrotranscribed DNA in CA-positive complexes. We adapted ANCHOR technology (NeoVirTech) (51, 52) to visualize HIV-1 DNA (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes

Blanco-Rodriguez

Gazi²,

Monel

et al. 2020

Preprint

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AbstractRetroviral replication proceeds through obligate integration of the viral DNA into the host genome. To enter the nucleus, the viral DNA must be led through the nuclear pore complex (NPC). During HIV-1 cytoplasmic journey, the viral core acts like a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for the reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core reshapes into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated to the pre-integration complex (PIC) during and after nuclear translocation, in particular, in human lymphocytes, the main target cells of HIV-1, is still enigmatic. In this study, we analysed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immuno-gold labelling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby uncovering the architecture of the potential HIV-1 PIC. Thus, we revealed DNA- and CA-positive complexes by correlated light- and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodelled in a “pearl necklace” shape. Thus, we observed how CA proteins reshape around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed by the integrase and the retrotranscribed DNA leads HIV-1 genome inside the host nucleus to potentially replicate.Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry.ImportanceHow the reverse transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. HIV-1 core has a size of ∼100 nm, largely exceeding that of the NPC channel (∼39 nm). Thus, a rearrangement of the viral CA proteins organization is required to achieve effective nuclear translocation. The mechanistic of this process remains undefined due to the lack of a technology capable to visualize potential CA sub-complexes in association with the viral DNA in the nucleus of HIV-1-infected cells.By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed in the outer and inner side of the NE, and they represent potential PIC.Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

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“…This histone deposition then promotes the initiation of transcription from the early viral gene promoters, before the transcriptional activator E1A is expressed. Live-imaging of incoming adenoviral genomes is also possible using eGFP-labeled SET [56] or direct labeling of incoming viral DNA [59]. Developing technologies to visualize viral genomes immediately after nuclear entry has been challenging, but detection of protein VII together with SET by immunofluorescence microscopy allows for tracking incoming viral DNA [56].…”

Section: Dna Entry Into the Nucleus And The Activation Of Viral Transmentioning

confidence: 99%

“…siRNAmediated knockdown of SET results in decreased initial viral transcription [44,45], which is thought to depend on the deposition of histones on incoming viral genomes. In these direct labeling experiments, known as ANCHOR3, the deletion of E1A and E3 allows infected cells to enter mitosis during which viral genomes associate tightly with the condensed host chromosomes [59]. Live-imaging of incoming adenoviral genomes is also possible using eGFP-labeled SET [56] or direct labeling of incoming viral DNA [59].…”

Section: Dna Entry Into the Nucleus And The Activation Of Viral Transmentioning

confidence: 99%

“…Live-imaging of incoming adenoviral genomes is also possible using eGFP-labeled SET [56] or direct labeling of incoming viral DNA [59]. In these direct labeling experiments, known as ANCHOR3, the deletion of E1A and E3 allows infected cells to enter mitosis during which viral genomes associate tightly with the condensed host chromosomes [59]. This observation suggests that incoming viral genomes are physically linked to host chromatin and may have implications in the context of persistent virus.…”

Section: Dna Entry Into the Nucleus And The Activation Of Viral Transmentioning

confidence: 99%

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Epigenetics and the dynamics of chromatin during adenovirus infections

et al. 2019

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Edited by Urs GreberAbbreviations ADP, adenovirus death protein; Ad5 or Ad 12, human adenovirus type 5 or 12, or other type referenced; BHK, baby hamster kidney; BrdU, bromodeoxyuridine; CTCF, CCCTC binding factor; DBS, double stranded break; DDR, DNA damage response; E1A, early 1 A; E4orf3 or E4orf6, early 4 open reading frame 3 or 6; HAdV-A12, human adenovirus subgroup A type 12, or different subgroup or type referenced; HMGB, high-mobility group box; hPaf1, human RNA polymerase II-associated factor 1; ISGs, interferon-stimulated genes; PML, promyelocytic leukemia; snRNP, small nuclear ribonuclear protein; SET or TAF-1, SE translocation or template activating factor; VRCs, viral replication centers.

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

Cited by 41 publications

References 66 publications

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes

Epigenetics and the dynamics of chromatin during adenovirus infections

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