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.

Fig. 1.

Recovery of transfected biotinylated DNA from HeLA cells with streptavidin. HeLa cells were transfected with a plasmid encoding pEGFP that was not modified and 16 h later with a biotinylated plasmid encoding pLacO (A). The cells were collected and incubated with streptavidin-coated beads as described in the text. Beads were rinsed extensively and subject to RT-qPCR. Primers were selected to amplify β-lactamase, β-actin, and EGFP. The β-lactamase sequence was unique to pLacO and served as a reference to determine the amount of biotinylated DNA in the sample. EGFP and β-actin were used to determine the amount of pEGFP and host chromatin, respectively (B).

Fig. 2.

Recovery of protein bound to biotinylated DNA. HEp-2 cells were either mock-treated or transfected with nonbiotinylated pLacO or biotinylated pLacO. Samples were collected as described in the text. Protein from samples was separated in an AU-gel, transferred to nitrocellulose, stained with Ponceau-S (A), or reacted with antibody directed against H3K9Me (B). Purified histones (lane 1) were included as a reference. Protein was present only in samples transfected with biotinylated DNA (lane 4) and form bands that aligned with the histones.

Fig. 3.

Knockdown of Ddx17 by siRNA leads to a decrease in the amount of viral mRNA and protein present at 6 h after infection. HEp-2 cells were transfected with 200 pmol of either control siRNA or siRNA targeting Ddx17 mRNA for depletion. Seventy-two hours after infection, monolayers were exposed to HSV-1(F) at a ratio of 1.0 pfu per cell. After 6 h after infection the cells were collected, and the RNA was purified and analyzed by RT-qPCR (A) or immunolotting (B). (C) The amounts of mRNAs encoding Ddx17, ICP0, ICP27, TK, ICP8, and US3 proteins. The results were normalized with respect to mRNA levels in control SI RNA-treated cells. (D) Panel depicts the amount of Ddx17, ICP8, and TK protein as determined by immunoblotting. Actin served as a loading control.

Fig. 4.

Accumulation of viral protein in cells mock-treated or transfected with control plasmid pcDNA, plasmid myc-Ddx17, or myc-Ddx17K50R. 293T cells were either mock treated (lanes 1, 2, and 6) or transfected with 1.5 μg of either control plasmid pcDNA (lanes 3 and 7), plasmid encoding myc-tagged Ddx17 (lanes 4 and 8), or plasmid encoding myc-tagged Ddx17 mutant K50R (lanes 5 and 9). Mutation K50R abolishes SUMOylation at residue 50. After 24 h, cells were either mock-treated (lane 1) or exposed to 1.0 pfu of HSV-1(F) per cell. Cells were collected at 4 h (lanes 2–5) and 6 h (6–9) after infection. Fifty micrograms of the proteins were analyzed by immunoblotting with antibodies directed against ICP0, ICP27, and myc. Actin served as a loading control.

Fig. 5.

Localization of Ddx17 in infected cells. The figure shows the localization of Ddx17 in HEp-2 cells fixed 4 h (A–F) and 6 h (G–I) after infection. The cells were also reacted with antibody directed against ICP0 (A–C) and ICP4 (D–I). As shown by the Inset in C, a portion of Ddx17 localizes to sites adjacent to and partially overlapping ICP0. The distribution of Ddx17 and ICP4 in cells fixed at 4 and 6 h after infection shows a portion of Ddx17 localized to viral transcription compartments (D–I).