Down-regulation of A Human Herpesvirus 1 (HHV-1) MicroRNA in Infected Cells by Goniothalamin Treatment

Goniothalamin (GTN) has been proven to cause cell cycle arrest and apoptosis in human herpesvirus 1 (HHV-1) infected cells, but interestingly our preliminary transcriptomic analysis revealed other possible modes of action. The data showed that GTN treatment of HHV-1 clinical strain infected cells induced expression of the KLHL24 gene that encodes the Kelch-like 24 protein (KLHL24), a transcriptional inhibitor of HHV-1 immediate-early and early genes. An miRNA, hsv1-miR-H27, produced by HHV-1 has also been discovered to control the expression of KLHL24. In order to understand the cause of KLHL24 up-regulation, a time point study was conducted to investigate the effect of GTN on KLHL24 and hsv1-miR-H27 expression. Through RT-qPCR analysis, we found that HHV-1 down-regulated KLHL24 significantly (p < 0.05) starting from 12 hpi, while a significant up-regulation (p < 0.05) was observed upon GTN treatment of the infected cells at 4 and 8 hpi. For protein level analysis, significant down-regulation of KLHL24 (p < 0.05) was observed at all time points in HHV-1 infected cells. Intriguingly, treatment with GTN on HHV-1 infected cells showed no significant changes in protein expression compared to cells without any treatment. In addition, the miRNA hsv1-miR-H27 was detected from 16 hpi and treatment with GTN on infected cells showed down-regulation of the miRNA. This was in congruity with the recovery of KLHL24 down-regulation in GTN treated HHV-1 infected cells, confirming that GTN caused down-regulation of hsv1-miR-H27 that governs the expression of KLHL24. This study provides insights that GTN could be a potential multifaceted antiviral. ImportanceThis study provides evidence that GTN possesses a distinct mode of antivirus against HHV-1 compared to currently available antivirals. Our findings showed that GTN caused the down-regulation of a viral miRNA, which inhibits the expression of a cellular protein known as KLHL24. This protein serves as a transcriptional inhibitor of HHV-1 immediate-early and early genes. The down-regulation of this miRNA lead to the up-regulation of KLHL24 and eventually halted HHV-1 replication. With the previously reported antiviral mechanism and the outcome of this study, GTN is a potential multifaceted anti-HHV-1 agent.