DNA introduced into cells by infection or transfection is immediately coated by repressive histones and repressors. Viruses have evolved mechanisms to derepress their genes. What we know of these events is based on ChIP analyses that help identify the proteins involved in these processes, provided their identity is suspected and reagents are available. This paper describes a method that enables identification of proteins not previously linked to these processes, based on the observation that HSV derepresses both viral DNA and DNAs introduced concurrently by transfection by using the transfected DNA as the surrogate for viral DNA.
Use of biotinylated plasmid DNA as a surrogate for HSV DNA to identify proteins that repress or activate viral gene expression. PNAS USA, 05 December 2012. doi: 10.1073/pnas.1218783109
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.
And also: I love the new Significance box on PNAS papers – similar to the Author Summary in PLOS ONE. I wish all journals would do this.