Effects on exocytosis by two HSV-1 mutants unable to block autophagy

Abstract

ABSTRACT Herpes simplex virus 1 (HSV-1) is a prominent human pathogen that manipulates host responses to support the infection. HSV-1 infection triggers an increased release of extracellular vesicles (EVs) through the CD63 biogenesis pathway that contains the DNA sensor STING and exerts an antiviral effect on recipient cells. However, it is currently unknown how different viral proteins influence EV production. The viral proteins ICP0 and ICP34.5 are important for blocking host responses, including autophagy. Considering that autophagy and exocytosis are interwoven processes regulating intercellular communication, we sought to determine changes in EV biogenesis after infection with a ΔICP0 and a ΔICP34.5 virus that displayed defects in blocking autophagy compared with wild-type virus. ΔICP34.5 virus induced a greater release of EVs as compared to HSV-1(F), whereas ΔICP0 virus did not. EVs released by ΔICP34.5-infected cells differ in origin from those released by HSV-1(F)-infected cells as they contained markers of microvesicles and apoptotic bodies, but lacked major tetraspanins (CD63 or CD81) or lysosomal membrane proteins. EVs released by ΔICP0-infected cells are generated through the CD63 biogenesis pathway activated by HSV-1(F) but displayed reduced amounts of cargo. EVs released by these mutant viruses triggered robust innate immunity gene expression in uninfected recipient cells that exceeded host responses due to infection by these viruses. Overall, we have characterized how infection with viruses unable to block autophagy affects EV biogenesis, and their effect on recipient cells. IMPORTANCE Pathogens often hijack extracellular vesicle (EV) biogenesis pathways for assembly, egress, and cell-to-cell spread. Herpes simplex virus 1 (HSV-1) infection stimulated EV biogenesis through a CD63 tetraspanin biogenesis pathway and these EVs activated antiviral responses in recipient cells restricting the infection. HSV-1 inhibits autophagy to evade the host, and increased CD63 exocytosis could be a coping mechanism, as CD63 is involved in both cargo delivery to lysosomes during autophagy and exocytosis. We analyzed exocytosis after infection with two HSV-1 mutants, a ΔICP34.5 and a ΔICP0, that could not inhibit autophagy. Unlike HSV-1(F), neither of these viruses stimulated increased EV biogenesis through the CD63 pathway. ΔICP34.5 stimulated production of microvesicles and apoptotic bodies that were CD63-negative, while ΔICP0 displayed an overall reduced production of EVs. These EVs activated innate immunity gene expression in recipient cells. Given the potential use of these mutants for therapeutic purposes, the immunomodulatory properties of EVs associated with them may be beneficial.