Circadian CLOCK histone acetyl transferase localizes at ND10 nuclear bodies and enables herpes simplex virus gene expression

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ICP0, a key herpes simplex virus regulatory protein, functions first in the nucleus and then in the cytoplasm. The duration of its nuclear sojourn in cells transfected with DNA and then infected is related to the quantity of transfected DNA. Furthermore, ICP0 transactivates both viral genes and genes encoded by the transfected DNA. The data support the hypothesis that ICP0 is retained in the nucleus until it completes the replacement of repressive chromatin with effector proteins that enable transcription of both DNA templates. To identify the effector proteins, we transfected cells with biotinylated DNA encoding a nonviral gene and then infected the cells with wild-type virus. Proteins bound to transfected biotinylated plasmid recovered from mock-treated and infected cells were identified using mass spectrometry followed by appropriate database search. The transfected DNA from mock-infected cells yielded proteins associated with repression, whereas DNA recovered from infected cells included proteins known to enable transcription and proteins that have not been previously associated with that role. To test the hypothesis that the proteins hitherto not known to associate with viral gene expression are nevertheless essential, we tested the role of the DEAD-box helicase Ddx17. We report that Ddx17 plays a critical role in the expression of early and late viral genes. Thus, biotinylated DNA recovered from transfected infected cells can function as a surrogate for viral DNA and is a rich source of proteins that play a role in viral gene expression but which have not been previously identified in that role. DNA binding proteins | gene derepressionU pon entry into the nucleus, the DNA of HSV-1 is immediately coated by repressive cellular proteins and bound to a dynamic nuclear body known as ND10 (1). Expression of viral genes requires sequential derepression at least at two checkpoints (2). In the first VP16, a viral tegument protein introduced into the cell during infection, recruits to the promoters of the α (immediate early) genes numerous host proteins that include the Octamer binding protein 1, Host cell factor 1, and Lysine Specific Demethylase 1 (LSD1) (1, 2). The consequence of derepression of the α genes is the synthesis of the six α proteins. One of these proteins, ICP0, plays a key role in the transition through the second checkpoint. Briefly, newly synthesized ICP0 colocalizes with ND10, recruits the ubiquitin-conjugating enzyme UbcH5a, and mediates the degradation of PML and SP100, key components of the ND10 nuclear bodies (3-5). In addition, it interacts with a repressor complex whose key components are HDAC-1/2, CoREST, LSD1, and REST. In this instance ICP0 binds to CoREST and dislodges HDAC-1/2 from the repressor complex (6-8). Last, ICP0 either recruits or enhances the recruitment of numerous host proteins to the viral replication compartment erected in the space formerly occupied by the ND10 nuclear bodies. These include cyclin D3, cdc34, CoREST, USP7, Bmal1, and the histone acetyl transferase CLOCK (...

Section: Discussionmentioning

confidence: 99%

Use of biotinylated plasmid DNA as a surrogate for HSV DNA to identify proteins that repress or activate viral gene expression

Mallon¹,

Wakim²,

Roizman³

2012

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“…Approximately 60 μg of proteins per sample was subjected to further analysis. Proteins were electrophoretically separated on 10% denaturing polyacrylamide gels, electrically transferred to nitrocellulose sheets, and reacted with the primary antibodies as described before (32). The rabbit polyclonal antibody to ICP0, the mouse monoclonal antibody to Us11 (Goodwin Institute for Cancer Research), and the rabbit polyclonal antibody to US3-PK were used in a dilution of 1:1,000.…”

Section: Methodsmentioning

confidence: 99%

“…The sources and procedures for propagation of HEp-2, HeLa, HEK293T (30,31), HEL, U2OS, and Vero cells are described elsewhere (32). The viruses used in this study were HSV-1(F), a limited passage HSV-1 prototype used in this laboratory (33); R7910 lacking both copies of the α0 gene encoding ICP0 (6); R7914, encoding ICP0 carrying the D199A substitution in ICP0 (24); R7041 lacking the US3-PK gene (34); R7356 lacking the UL13 gene (35); the RF mutant derived from the HSV-1(KOS) strain and carrying the C116G/C156A codon substitutions in the ring finger domain of ICP0 (36); and the d120 mutant derived from the KOS strain and lacking both copies of the ICP4 gene (23).…”

Section: Methodsmentioning

confidence: 99%

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HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation

Kalamvoki

Roizman

2014

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STING (stimulator of IFN genes) activates the IFN pathway in response to cytosolic DNA. Knockout of STING in mice was reported to exacerbate the pathogenicity of herpes simplex virus 1 (HSV-1). Here we report the following: (i) STING is stable in cancer-derived HEp-2 or HeLa cells infected with wild-type HSV-1 but is degraded in cells infected with mutants lacking the genes encoding functional infected cell protein 0 (ICP0), ICP4, or the US3 protein kinase (US3-PK). In HEp-2 cells, depletion of STING by shRNA results in a decrease in the yields of wild-type or ΔICP0 viruses. (ii) STING is stable throughout infection with either wild-type or ICP0 mutant viruses in human embryonic lung cells (HEL) or HEK293T cells derived from normal tissues. In these cells, depletion of STING results in higher yields of both wild-type and ΔICP0 viruses. (iii) The US3-PK is also required for stabilization of IFI16, a nuclear DNA sensor. However, the stability of IFI16 does not correlate positively or negatively with that of STING. IFI16 is stable in STING-depleted HEL cells infected with wild-type virus. In contrast to HEL cells, IFI16 was undetectable in STING-depleted HEp-2 cells, and hence the role of HSV-1 in maintaining IFI16 could not be ascertained. The results indicate that in HSV-1-infected cells the stability of IFI16 and the function and stability of STING are dependent on cell derivation, the functional integrity of ICP0, and US3-PK, an indication that in wild-type virus-infected cells both proteins are actively stabilized. In HEp-2 cells, the stability of IFI16 requires STING.innate immunity | cell transformation T he studies described in this report stem from two observations. First, a voluminous literature singles out the infected cell protein 0 (ICP0) as the major herpes simplex virus 1 (HSV-1) protein dedicated to defeating host responses to infection (1). Many of the functions of ICP0 designed to defeat host responses are executed by direct interaction between ICP0 and host proteins (2-8). In some instances, a plausible connection is apparent but no physical contact between ICP0 and the host effector protein is demonstrable (9, 10). Thus, ICP0 is associated with blocking the activation of IRF3 although a physical interaction between ICP0 and IRF3 has not been reported (10). One hypothesis that could explain such observations is that ICP0 interacts with the partners of the targeted protein rather than the target itself.Second, ICP0 accumulates during the first phase of the replicative cycle in the nucleus in which it performs multiple functions to enable efficient replication. Sometime between 5 and 9 h after exposure of cells to virus and depending on cell line, functional integrity of ICP0, and the amount of foreign DNA introduced into the cell, etc., ICP0 disappears from the nucleus and accumulates in the cytoplasm (11, 12). Several functions of ICP0 linked to physical interactions with cytoplasmic proteins have been described. Thus, ICP0 interacts with EF-1δ and enhances translation efficiency and with CIN85 t...

“…ICP0 is a major multifunctional protein created immediately after infection. Among its major functions are the recruitment of CLOCK histone deacetylase to the viral transcriptome (40), the dissociation of the CoREST/REST/LSD1 repressor complex from its cognate sites on viral DNA (41,42) and the degradation of PML and SP100 (11,15,43). ΔICP0 mutants replicate in U2OS cells but poorly in numerous cell lines, including human (e.g., HEp-2) and African green monkey kidney (Vero) cells (44)(45)(46).…”

Section: At a Low Ratio Of Virus Per Cell δIcp0 Virus Replicates To mentioning

confidence: 99%

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments

Roizman

2017

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Nuclear domain 10 (ND10) bodies are small (0.1–1 μM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-β. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-β in PML−/− cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML−/− cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML−/− cells, SP100−/− cells retain the sensitivity of parental SP100+/+ cells to IFN-β and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100−/− cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100−/− cells than in parental SP100+/+ cells or in PML−/− cells.