RESUMO
We reported that gamma-hydroxybutyrate (GHB) is released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. However, the cellular biochemical processes involved in the production of GHB in infected cells are unclear. This study aims to shed light on the biochemical pathway and the stage within the viral life cycle responsible for the release of GHB in infected cells. UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) treatments were used to inhibit HSV-1 replication at various stages. Vero cells treated with UV-inactivated HSV-1 significantly decreased GHB production. However, ACV or CHX treatments did not affect GHB production. We also showed that inhibition of glycolytic enzyme enolase by sodium fluoride (NaF) significantly reduces GHB production upon infection. This finding suggests that suppression of glycolytic activity negatively affects cellular GHB production. Our data also indicated that succinic semialdehyde dehydrogenase, an enzyme involved in the shunt of the tricarboxylic acid (TCA) cycle to generate succinic acid, was decreased upon infection, suggesting that infection may trigger the accumulation of succinic semialdehyde, causing the production of GHB. Although the precise mechanism has yet to be defined, our results suggest that early events following infection modulates the release of GHB, which is generated through the metabolic pathways of glycolysis and TCA cycle.
RESUMO
Volatile organic compounds (VOCs) release triggered by infection of DNA virus has not been studied extensively. Previously, we reported that gamma-butyrolactone (GBL), a VOC, was released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. Based on the metabolic pathway and chemical conversion of GBL, we hypothesized that infected cells produce gamma-Hydroxybutyric acid (GHB) as a key pathway intermediate for the subsequent production of GBL. An analytical technique for the rapid detection of GHB is crucial for further understanding its role in the cellular response to HSV-1 infection. To address this, we developed a sensitive, reliable, and specific method for the detection and quantification of GHB in mammalian cell culture using a pre-column derivatization approach. Our data showed that the carboxylic acid functional group of GHB could be derivatized with 3-nitrophenylhydrazine hydrochloride (3-NPH) to produce its hydrazineyl derivative. Unlike GHB, the derivative could be detected seamlessly in HPLC-MS. We also demonstrate quantitive conversion of GHB into the derivative with over 95% yield at a range of 1 µg/mL- 6 µg/mL GHB concentration. This method offers a rapid quantification of GHB in aqueous mixtures, especially in cultured extracts.