HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and Centromeres Regulate LAT Locus Transcription during Latency in Neurons

University of Glasgow, United Kingdom
PLoS Pathogens (Impact Factor: 7.56). 08/2012; 8(8):e1002852. DOI: 10.1371/journal.ppat.1002852
Source: PubMed


Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called "Single" and "Multiple", which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. "Single" and "Multiple" patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the "Multiple" pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction.

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Available from: Patrick Lomonte, Feb 14, 2014
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    • "Promyelocytic leukemia nuclear bodies are known to perform multiple roles that vary when cells are infected with different viruses. For example, in neuronal cells infected with herpes viruses, such as herpes simplex virus type 1 and varicella zoster virus, the viral genomic DNA or the nucleocapsids were detected inside large PML-NBs, suggesting that PML-NBs are involved in herpes simplex virus type 1 viral latency control (39) or serve as an intrinsic antiviral host defense response against varicella zoster virus infection (40). In contrast, JC virus lytic infection indicated structural roles for PML-NBs in providing scaffolding for viral progeny production, but depletion of the pml gene from host cells in in vitro experiments did not dramatically affect the replication of JC virus, BK virus, or polyomavirus; therefore, PML-NBs would not be essential for viral replication (36, 37, 41). "
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    ABSTRACT: In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
    04/2014; 73(5). DOI:10.1097/NEN.0000000000000066
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    • "Reactivation of the virus occurs following different stresses, and ICP0 has been shown to be essential for full reactivation in a mouse model [26], [27]. Using a fluorescence in situ hybridization (FISH) approach specifically developed to detect latent HSV-1 genomes within neuronal tissues, we have shown that HSV-1 genomes are not randomly distributed in the nuclei of infected neurons [78]. Interestingly, a significant proportion of the infected neurons displayed HSV-1 genomes that co-localized with centromeres [78]. "
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    ABSTRACT: The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1). As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs), namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR). The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated) and CAD (CENP-A Distal) complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase) digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs), we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.
    PLoS ONE 09/2012; 7(9):e44227. DOI:10.1371/journal.pone.0044227 · 3.23 Impact Factor
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    ABSTRACT: Upon entry of the viral genome into the nucleus, herpes simplex virus type 1 (HSV-1) gene expression is rapidly repressed by constitutively expressed cellular proteins. This intrinsic antiviral defence is normally counteracted by ICP0, which allows virus infection to proceed efficiently. Replication of ICP0-null mutant HSV-1, however, is severely repressed by mechanisms that are conferred, at least in part, by nuclear domain 10 (ND10) components including hDaxx, PML and Sp100. To investigate if these ND10 components repress viral gene expression in a cooperative manner, we simultaneously depleted host cells for hDaxx, PML and Sp100 by multiple shRNA knock-down from a single lentivirus vector. We found that replication and gene expression of ICP0 null mutant HSV-1 were cooperatively repressed by hDaxx, PML and Sp100 immediately upon infection, and all stages of virus replication were inhibited. Plaque forming efficiency was enhanced at least 50-fold in the triple-depleted cells, a much larger increase than achieved by depletion of any single ND10 protein. Similar effects were also observed during infection of triple depleted cells with human cytomegalovirus (HCMV). Moreover, using a cell culture model of quiescent infection, we found that triple depletion resulted in a much higher number of viral genomes escaping repression. However, triple depletion was unable to fully overcome the ICP0-null phenotype, implying the presence of additional repressive host factors, possibly components of the SUMO-modification or DNA repair pathways. We conclude that several ND10 components cooperate in an additive manner to regulate HSV-1 and HCMV infection.
    Journal of Virology 12/2012; 87(4). DOI:10.1128/JVI.02950-12 · 4.44 Impact Factor
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