Bacteria-herpesvirus interaction reveals a proviral role of ISGylation in promoting herpesvirus capsid assembly

Herpesviruses are common pathogens of the oral cavity, yet how they interact with other oral microbes are poorly understood. Using murine gamma-herpesvirus 68 (MHV68) as a model for human gamma-herpesviruses, we discover that ISGylation is hijacked to facilitate viral capsid assembly and lytic replication. Co-infection with the oral <i>Aggregatibacter actinomycetemcomitans</i> ( <i>A.a</i> .) and MHV68 synergistically induced interferon-stimulated gene 15 (ISG15) expression and global ISGylation. Proteomic profiling revealed viral structural proteins as the dominant targets of ISGylation in MHV68-infected cells. Genetic ablation of ISGylation, using ISGylation-resistant recombinant MHV68 and ISG15-deficient mouse embryonic fibroblasts (MEFs), demonstrated that ISGylation of the major capsid protein ORF25 is required for efficient capsid assembly and maturation into the infectious C-type virions. Strikingly, introduction of a <i>de novo</i> ISGylation site into the ISGylation-resistant MHV68 was sufficient to restore ISGylation, capsid assembly, and virion maturation. Consistent with these findings, <i>A.a.</i> failed to enhance MHV68 lytic replication in ISG15-deficient MEFs. Extending this mechanism to representative herpesviruses, we showed that ISGylation of major capsid proteins is broadly required for efficient lytic replication. Together, these findings uncover a previously unrecognized strategy by which herpesviruses exploit an ISG15 innate immune effector, amplified by microbial co-infection, to promote virion biogenesis and productive infection.