Abstract
ABSTRACTHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate dynamic interactions between herpes simplex virus type 1 (HSV-1) DNA and viral and host proteins throughout infection, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. To accomplish this, virus stocks were prepared in the presence of ethynyl-modified nucleotides to enable covalent tagging of viral genomes after infection for analysis of viral genome-protein interactions by imaging or affinity purification. Affinity purification was combined with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry to enable the distinction between proteins that were brought into the cell by the virus or expressed within the infected cell before or during infection. We found that input viral DNA progressed within 6 h through four temporal stages where the genomes sequentially (i) interacted with intrinsic antiviral and DNA damage response proteins, (ii) underwent a robust transcriptional switch mediated largely by ICP4, (iii) engaged in replication, repair, and continued transcription, and then (iv) transitioned to a more transcriptionally inert state engaging de novo-synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on input genome-proteome dynamics.
Highlights
Herpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host
Transcription of immediate early (IE) viral genes is activated by the viral tegument protein VP16 [8, 9], transcription of early and late viral genes is activated by the IE gene product ICP4 [10,11,12], and transcription of late viral genes is coupled to viral DNA replication by an unknown mechanism
To examine events that occur on viral genomes, we previously developed an approach based on isolation of proteins on nascent DNA [13] to selectively label replicating viral DNA within infected cells with ethynyl-modified nucleotides (5-ethynyl-2=-deoxycytidine [EdC] or 5-ethynyl-2=-deoxyuridine [EdU]) to enable the covalent conjugation to biotin-azide or Alexa Fluor-azide [14]
Summary
Herpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. Herpesviruses are a family of highly prevalent eukaryotic viruses that share strong evolutionary relationships with their hosts [1] They have developed sophisticated mechanisms to invade host cells, alter cellular activities, and redirect host factors for use in viral processes. Initial productive infection occurs in epithelial cells where the viral genome is prolifically transcribed and replicated, resulting in many new virus progeny. Nuclear stages of productive infection involve coordinated events occurring on the viral genome, which begin with the transfer of viral DNA into the host nucleus through the nuclear pore shortly after infection [3]. Replicated DNA is packaged into preassembled capsids, which subsequently exit the nucleus How these events are staged with respect to the actions of viral and cellular protein complexes acting on the viral genome is unclear. This is mostly due to issues of sensitivity in the measurements of processes occurring during single step growth, as well as the fact that time of occurrence of crucial events can be obscured by events that are more quantitatively robust
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