Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes and which variant forms of the core histone proteins are deposited are incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histones H3.1 and H3.2 (collectively, H3.1/2) and H3.3 during lytic and latent HCMV infections. Here, we show that they are deposited on HCMV genomes during lytic and latent infections, suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating that both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections. IMPORTANCEIt is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways and viral fate determinations (lytic or latent) and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how the genomes are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing that HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to posttranslational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.T he large genomes of eukaryotic cells must be highly compacted in order to fit within the restricted volume of the nucleus. To accomplish this, eukaryotic DNA is packaged into a repeating nucleoprotein structure known as chromatin, the basic subunit of which is the nucleosome (1, 2). The core nucleosome particle consists of approximately 146 bp of DNA wrapped nearly twice around a histone hetero-octamer consisting of two copies each of the four core histone proteins H2A, H2B, H3, and H4 (3). Nucleosomes are further compacted into higher-order chromatin fibers through the incorporation of the linker histone H1, other nonhistone proteins, and structural RNA components (4-6).Wrapping DNA in nucleoso...

Section: Discussionmentioning

confidence: 96%

Canonical and Variant Forms of Histone H3 Are Deposited onto the Human Cytomegalovirus Genome during Lytic and Latent Infections

Albright

Kalejta

2016

“…In the U2OS cell, ATRX is not expressed due to the deletion of the gene (37). Recently it has been reported that ATRX functions together with Daxx as a negative regulator in Ad gene expression and that the expression of EGFPtagged ATRX can reconstitute the functional Daxx/ATRX complex in U2OS cells (38). Therefore, U2OS cells were first transfected with the expression vectors for EGFP alone or EGFP-ATRX and then infected with Ad5-GFP and subjected to IF analyses.…”

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

confidence: 99%

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

Komatsu

Nagata

Wodrich

2016

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.

“…Furthermore, we employed Daxx-depleted U2OS cells (78) to additionally diminish the repressive effect of the Daxx-ATRX chromatin remodeling complex. We observed that Sp100A stimulated transcription from the Ad promoters ϳ2-to 3-fold in both cell lines.…”

Section: Sp100 Depletion Promotes Ad Progeny Production and Early Virmentioning

confidence: 99%

Sp100 Isoform-Specific Regulation of Human Adenovirus 5 Gene Expression

Berscheminski

Wimmer

Brun

et al. 2014

Self Cite

Promyelocytic leukemia nuclear bodies (PML-NBs) are nuclear structures that accumulate intrinsic host factors to restrict viral infections. To ensure viral replication, these must be limited by expression of viral early regulatory proteins that functionally inhibit PML-NB-associated antiviral effects. To benefit from the activating capabilities of Sp100A and simultaneously limit repression by Sp100B, -C, and -HMG, adenoviruses (Ads) employ several features to selectively and individually target these isoforms. Ads induce relocalization of Sp100B, -C, and -HMG from PML-NBs prior to association with viral replication centers. In contrast, Sp100A is kept at the PML tracks that surround the newly formed viral replication centers as designated sites of active transcription. We concluded that the host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression. Ad-dependent loss of Sp100 SUMOylation is another crucial part of the virus repertoire to counteract intrinsic immunity by circumventing Sp100 association with HP1, therefore limiting chromatin condensation. We provide evidence that Ad selectively counteracts antiviral responses and, at the same time, benefits from PML-NB-associated components which support viral gene expression by actively recruiting them to PML track-like structures. Our findings provide insights into novel strategies for manipulating transcriptional regulation to either inactivate or amplify viral gene expression. IMPORTANCEWe describe an adenoviral evasion strategy that involves isoform-specific and active manipulation of the PML-associated restriction factor Sp100. Recently, we reported that the adenoviral transactivator E1A targets PML-II to efficiently activate viral transcription. In contrast, the PML-associated proteins Daxx and ATRX are inhibited by early viral factors. We show that this concept is more intricate and significant than originally believed, since adenoviruses apparently take advantage of specific PML-NBassociated proteins and simultaneously inhibit antiviral measures to maintain the viral infectious program. Specifically, we observed Ad-induced relocalization of the Sp100 isoforms B, C, and HMG from PML-NBs juxtaposed with viral replication centers. In contrast, Sp100A is retained at Ad-induced PML tracks that surround the newly formed viral replication centers, acting as designated sites of active transcription. The host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression.