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

Abstract

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 (...

Proteomic analysis of the cullin 4B interactome using proximity‐dependent biotinylation in living cells

“…This marked reduced expression was confirmed by Western blot (shown in Fig. C), consistent with previous biochemical data .…”

Integrative Interactome and Ubiquitinome Analyses Reveal Multiple Regulatory Pathways Targeted by ICP0 in HSV-1 Infected Neuronal Cells

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