The Role of MSK1 in EBV Genome Maintenance

• Main Authors: Hsin Kuo, 郭馨
• Other Authors: Chi-Ju Chen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/vdxm93

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 106 === Epstein-Barr virus (EBV) is a human γ-herpesvirus and more than 90 % people are infected by EBV. EBV establishes latent infection in B cells. EBV can be reactivated to undergo lytic replication through stimulating B cell receptor signaling. Maintaining EBV genome in cells is important to EBV latent infection. In our previous study, MSK1 was found playing a role in EBV latent infected Burkitt’s lymphoma cells, Akata (+). Using lentivirus infection to express shRNA targeting MSK1 (shMSK1#14), we found EBV genome was lost. To further confirm this phenomenon, I repeated knockdown of MSK1 in Akata (+) cells using shMSK1#14 expressing lentivirus. Results show that about one-month later EBV genome started to lose. However, I found EBV was reactivated starting at 12 days after lentivirus infection accompanying increase of EBV genome, which affected me to observe the amounts of EBV episome. Therefore, I established a Tet-On system to express shMSK1#14 (TetO shMSK1#14) to improve this condition. And found Doxycycline was able to inhibit MSK1 expression effectively. The amounts of EBV genome were not different between -Dox and +Dox cells though. We speculate that it was because lentivirus infection induced EBV reactivation causing an increase of EBV genome. I confirmed this speculation by experiments afterwards. In order to inhibit genome replication which interferes with the observation intent, I used phosphonoacetic acid (PAA), a replication inhibitor. Treatment of PAA was able to inhibit EBV DNA replication upon spontaneous reactivation. It rewards to be seen if PAA treatment can be useful in this study. It is known that EBNA1 protein and origin of replication oriP are important to maintain latent EBV genome in cells. To study whether the mechanism of inhibition of MSK1 leading to lost EBV genome is through the regulation of EBNA1 and oriP, I chose a plasmid pRep10-EGFP, which expressed Gly/Ala truncated EBNA1 and oriP. The plasmid was intended for me to observe the relationship between EBNA1/oriP and MSK1-dependent EBV genome maintenance. Because of the condition of 293T shMSK1#14 cells were poor after pRep10-EGFP transfection and hygromycin B selection, was not able to use this system in the subsequent experiments.