- 學位論文
EB病毒BALF3於病毒生活史與鼻咽癌發生之角色探討
Roles of Epstein-Barr Virus BALF3 in Viral Life Cycle and Nasopharyngeal Carcinogenesis
摘要
EB病毒BALF3與第一型疱疹單純病毒溶裂複製有關的末端酶UL28具高度同源性，然而BALF3對於EB病毒生活史與宿主細胞的影響至今仍尚未清楚。為了釐清BALF3的特性，利用純化的BALF3蛋白來進行試管中酵素反應實驗，發現BALF3具有核酸內切酶的活性且可由鎂錳離子與ATP分子來調控。此外，當帶有EB病毒的鼻咽癌細胞中的EB病毒進入溶裂期時，BALF3會開始表現並由細胞質進入細胞核中。進一步將BALF3基因默化則明顯降低病毒DNA包裝作用與病毒顆粒的產生，同時也減少病毒DNA合成效率。另一方面，BALF3可造成EB病毒陰性的鼻咽癌細胞產生基因不穩定性如微核形成及DNA斷裂的增加，而細胞經過BALF3反覆表現影響後，出現顯著的基因拷貝數變異及細胞移行、細胞侵襲與球狀體形成等腫瘤特徵，同時將其細胞注射至具免疫缺陷的小鼠體中，與控制組相較之下則形成較大的腫瘤。最後，利用RNA微陣列發現BALF3反覆表現會影響許多癌症相關基因及致癌基因的表現，而其變化與基因變異相關。因此，由以上的實驗結果可得知EB病毒BALF3參與病毒DNA合成、切割與包裝進而影響病毒顆粒產生，以及也扮演EB病毒感染導致鼻咽癌發生的潛在角色。
並列摘要
Epstein-Barr virus (EBV) BALF3 is a homologue of the herpes simplex virus type 1 terminase UL28 that is responsible for viral lytic replication. However, the effects of BALF3 on the EBV life cycle and host cells are still unclear. In order to characterize BALF3, the purified protein was produced and examined in an enzymatic reaction in vitro, which determined that BALF3 acts as an endonuclease, and its activity is modulated by Mg2+, Mn2+, and ATP. Moreover, in EBV-positive nasopharyngeal carcinoma (NPC) cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following lytic induction, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Meanwhile, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the other hand, BALF3 has the ability to induce host genomic instability, such as micronucleus formation and DNA strand breaks. After recurrent expression of BALF3 in EBV-negative NPC cells, genomic copy number aberrations had accumulated to a significant extent, and tumorigenic features such as cell migration, cell invasion, and spheroid formation increased with the rounds of induction. In parallel experiments, cells after highly recurrent induction developed into larger tumor nodules than control cells when inoculated into NOD/SCID mice. Furthermore, RNA microarrays showed that differential expression of multiple cancer capability-related genes and oncogenes increased with recurrent BALF3 expression, and these changes were correlated with genetic aberrations. Therefore, EBV BALF3 is involved simultaneously in DNA synthesis, cleavage, and packaging, which accounts for the production of mature virions, and it also is a potential factor that mediates the impact of EBV on nasopharyngeal carcinogenesis.