# Phosphoproteome Remodeling upon CDK1 Inhibition Restricts HSV-1 IE Gene Transcription and Replication

## TL;DR

Inhibiting CDK1 disrupts HSV-1 replication by altering cell phosphorylation and reducing viral gene expression.

## Contribution

The study reveals CDK1's role in HSV-1 replication through phosphoproteome analysis and identifies key host factors.

## Key findings

- CDK1 inhibition reduces HSV-1 replication by 1000-fold and decreases IE mRNA and protein levels.
- Over 5500 phosphopeptides were identified, linking CDK1 activity to RNA polymerase II phosphorylation.
- Phosphorylation of POLR2A is significantly reduced with CDK1 inhibition during HSV-1 infection.

## Abstract

Cyclin-dependent kinase 1 (CDK1) regulates multiple cellular processes that HSV-1 can exploit to promote its own replication, particularly during the early steps of lytic infection. We investigated whether CDK1 inhibition disrupts immediate-early (IE) gene expression and analyzed the host phosphoproteome early in infection to identify putative host factors and mechanisms that facilitate HSV-1 IE gene expression and are controlled by CDK1. Human foreskin fibroblasts (HFFs) were pre-treated with a CDK1 inhibitor and showed a 1000-fold reduction in HSV-1 replication and significant reductions in IE mRNAs and protein levels at 4 hpi. We characterized cells after CDK1 inhibition and HSV-1 infection at 3 hpi by tandem mass spectrometry and identified >5500 phosphopetides (~2600 proteins), analyzing differential phosphorylation and protein–protein interactions. We validated CDK1 inhibition by detecting phosphorylation-specific decreases in known CDK1 substrates, as well as Robust Kinase Activity Inference. Rank- and network-based analyses of our dataset highlighted several candidate proteins, linking their CDK-directed phosphorylation to HSV-1 IE gene expression. Notably, the C-terminal domain of the large subunit of RNA polymerase II (RNAPII), POLR2A, is extensively phosphorylated, and its phosphorylation is significantly reduced upon CDK1 inhibition during viral infection. Taken together, these data support a model in which CDK1 activity maintains a transcriptionally permissive cellular state required for efficient HSV-1 IE gene expression. Our data suggest that when CDK1 is pharmacologically inhibited, key transcriptional facilitators are dysregulated, impairing viral transcription and replication.