HSV-1 temporally orchestrates RAP80 ubiquitination to balance viral replication and evasion [RNA-seq]

The mechanisms governing the complete replication cycle of herpes simplex virus type 1 (HSV-1) remain incompletely elucidated. Here, we showed that HSV-1 employs a biphasic strategy to dynamically regulate the host protein RAP80 through stage-specific ubiquitination, thereby coordinating its replication cycle. Upon entry, RAP80 undergoes phase separation to form antiviral condensates that encapsulate the viral genome and suppress viral transcription. The HSV-1-encoded E3 ubiquitin ligase ICP0 counteracts this antiviral mechanism by mediating RAP80 ubiquitination to dissolve these condensates, facilitating viral replication. In middle stage, RAP80 depletion leads to accumulation of R-loops and activation of the cGAS–STING–apoptosis axis, threatening viral persistence. In response, in middle and late stage, the viral deubiquitinase UL36USP deubiquitinates and stabilizes RAP80 to suppress R-loop accumulation and this immune-triggered apoptosis. This biphasic regulation of a single host factor represents a sophisticated viral strategy to ensure productive replication and dissemination. Notably, pharmacological disruption of RAP80 ubiquitination using engineered inhibitory peptides potently inhibits HSV-1 replication in vitro and in vivo. Furthermore, RAP80 inhibition enhances both chemosensitivity and oncolytic virotherapy in tumors. These findings reveal a ubiquitination-mediated host–pathogen equilibrium with therapeutic potential in antiviral and anticancer contexts.