Abstract
Endocytosis and exocytosis pathways are critical for the survival of the cells. Viruses often hijack these trafficking pathways to support cell entry, replication, and egress and to evade host antiviral responses. The infected cell protein 0 (ICP0) is an immediate early protein of alphaherpesviruses also known as the promiscuous transactivator. Most of the ICP0 functions are performed in the nucleus; however, ICP0 translocates to the cytoplasm after enabling viral gene expression where very little is known about its cytoplasmic roles. ICP0 interacts with the Cbl-interacting protein of 85 kDa (CIN85), a scaffolding protein for factors involved in endocytosis and protein sorting. The ICP0/CIN85 complex promotes endocytosis of surface receptors, including the epidermal growth factor receptor (EGFR) to suppress signaling and the virus entry receptor nectin-1 to ensure virus spread in uninfected cells. CIN85 recognizes consensus motifs Px(P/A)xxR in its binding partners, and two such motifs are stretched between ICP0 246-258 aa. Deletion of the CIN85-binding motifs of ICP0 disrupts its interaction with CIN85 and the localization of ICP0 to CIN85 vesicle-like structures. This ICP0 deletion further disrupts exocytosis of cargo associated with the CIN85 structures through extracellular vesicles (EVs), including autophagy and innate immune factors. Consequently, this ICP0 mutant virus fails to counteract antiviral responses and displays decreased progeny virus production. Overall, these studies have uncovered a novel mechanism by which HSV evades host antiviral responses through subjugation of endosomal and exocytosis pathways.IMPORTANCEHerpes simplex virus persists lifelong. For a successful infection, the virus has evolved different mechanisms, often complementary and redundant, to evade host defense pathways, highlighting that viral proteins are multifunctional. Multifunctional proteins display a variety of features, including different localization patterns, post-translational modifications, and ability to interact with different factors. The infected cell protein 0 (ICP0) of the virus encompasses three main functions; it is a promiscuous transactivator, an E3 ubiquitin ligase, and an inhibitor of DNA repressor complexes. The protein localizes to the nucleus early in infection and translocates to the cytoplasm following virus replication. In the cytoplasm, ICP0 interacts with the Cbl-interacting protein of 85 kDa (CIN85), an endocytosis adaptor, promoting surface receptor endocytosis, cargo sorting, and exocytosis. Innate immunity and autophagy-related factors are also found to be exocytosed via this pathway. This likely represents a novel immunoevasion function of HSV-1 ICP0 to suppress antiviral signaling.