HSV-1 US3 Hijacks Conserved Actin Regulatory Complexes to Drive F-Actin Remodeling

The herpes simplex virus 1 (HSV-1) US3 is a multifunctional serine/threonine kinase that promotes HSV-1 replication and spread. But its role and the mechanisms by which US3 regulates actin cytoskeletal remodeling remain poorly defined. We combined flow cytometry, confocal microscopy, immunoprecipitation-mass spectrometry (IP-MS), protein complex mapping, and machine learning to characterize US3-mediated F-actin dynamics. Flow cytometry and confocal microscopy showed that wild-type HSV-1 induces significant F-actin remodeling, while the ΔUS3 mutant displays F-actin levels comparable to uninfected cells, identifying US3 as a key regulator. IP-MS identified 47 high-confidence US3 interactors enriched in conserved actin regulatory complexes, including Arp2/3 nucleation machinery, formin-associated assemblies, cofilin severing complexes, and Rho-family GTPase modules. Mapping interactors to the CORUM database revealed clustering within actin nucleation, polymerization, and severing complexes, indicating that US3 operates through organized cellular machines. Machine-learning classifiers trained on label-independent mass-spectrometry features were used to prioritize interactors resembling known actin regulators; under protein-group-aware cross-validation, logistic regression performed best (average precision 0.24; ROC-AUC 0.66), and the analysis was interpreted as prioritization rather than de novo discovery. Pharmacological inhibition of Arp2/3 and formin pathways significantly attenuated US3-dependent F-actin remodeling, supporting the functional involvement of these pathways. Together, these findings are consistent with an inferred hierarchical axis in which US3 modulates Rho GTPase signaling and cofilin activation to promote F-actin disassembly, coordinating cytoskeletal remodeling required for efficient viral egress and spread.