Extracellular release of cytotoxic aggregates from HSV-1-replicating SH-SY5Y cells: involvement of alpha-synuclein and poly-ubiquitin conjugates.

Herpes simplex virus 1 (HSV-1) is a neurotropic enveloped DNA virus that latently infects sensory ganglia cell bodies. HSV-1 causes cold sores, keratitis, meningitis, and encephalitis. During the HSV-1-replicating phase (lytic phase), viral proteins and genomes are produced in the host cell and assemble into virions. Mature virions egress from the cell, leading to cell lysis. Here, we show that HSV-1-replicating neuroblastoma cells generate cytotoxic aggregates that are detected in the extracellular medium. We initially examined the intracellular and extracellular levels of polyubiquitin conjugates in SH-SY5Y cells undergoing HSV-1 replication. The dynamics of ubiquitination were altered within cells during the lytic phase. The accumulation of poly-ubiquitin conjugates and HSV-1 membrane proteins (gB and gD) was mainly detected at the juxtanuclear cytoplasm in HSV-1-replicating cells. Moreover, poly-ubiquitin conjugates, HSP90, gB, gD, alpha-synuclein, and cytotoxic molecules were detected in the culture supernatant. Co-immunoprecipitation assays revealed that polyubiquitin conjugates in the supernatant contained aggregated gD and alpha-synuclein. Furthermore, poly-ubiquitin conjugates and alpha-synuclein in the culture supernatant contributed to its cytotoxic effects. Our results indicate that HSV-1-replicating SH-SY5Y cells produce not only virions but also cytotoxic protein aggregates containing poly-ubiquitin conjugates and alpha-synuclein. These findings may provide insight into mechanisms underlying virus-induced protein aggregation and cytotoxicity, which may be relevant to neurodegenerative processes.