Following productive, lytic infection in mucosal epithelial cells located at the portal of entry in the body, herpes simplex virus-1 (HSV-1) establishes a lifelong, silent infection in sensory neurons. The virus is occasionally reactivated due to weakened immune response or stress causing diseases that range in severity from benign cold sores to encephalitis. HSV-1 contributes to exacerbation of neurodegenerative diseases such as Alzheimer's and facilitates infection by other pathogens such as HIV-1. Extracellular vesicles (EVs) are released by all types of cells, as they constitute a major mechanism of intercellular communication, and they can influence recipient cell functions. During HSV-1 infection two populations of EVs are readily detectable. The first population is produced through a tetraspanins biogenesis pathway. These are the most abundant EVs released from HSV-1 infected cells, which are enriched in CD63 tetraspanin and carry host factors such as the STimulator of INterferon Genes (STING), a sensor of DNA in the cytosol. These CD63+ EVs can activate innate immune responses in uninfected recipient cells and suppress a subsequent HSV-1 infection. The second population of EVs is produced through the endosomal sorting complex required for the transport (ESCRT) pathway, thus we named them ESCRT+ EVs. These EVs carry ESCRT components along with selected viral tegument and envelope protein and they appear to have a proviral role. In addition to these two population of EVs, earlier studies have reported the production of capsidless particles during HSV-1 infection composed of tegument and envelope proteins but lacking viral genome. These particles are lighter than HSV-1 virions and are known as L-particles. L-particles resemble the ESCRT+ EVs in that they carry viral proteins and have a proviral effect but are denser than ESCRT+ EVs and enriched in viral proteins. We hypothesize that distinct populations of EVs are released during HSV- 1 infection with different effects on the infection. To test our hypothesis, we have formulated three Aims. In Aim 1, we propose to compare the cargo of ESCRT+ EVs and CD63+ EVs with L-particles produced during HSV-1 infection using a mass spectrometry approach. The results of this analysis will generate important information about messages communicated by infected cells. In Aim 2, we propose to determine EV biogenesis pathways involved in the production of ESCRT+ EVs, CD63+ EVs and L-particles during HSV-1 infection. For this, we will use a gene silencing approach to compromise selected EV biogenesis pathways or we will use mutant viruses to determine the impact of selected viral genes on EV biogenesis. In Aim 3, we propose to determine the type of responses that CD63+ EVs, ESCRT+ EVs and L-particles trigger in recipient cells and determine the consequences to the infection. These studies will provide important information on the biogenesis, cargo, and functions of EVs released by HSV-1 infected cells, which are essential to determine their impact on HSV-1 pathogenesis.