EB病毒Rta蛋白質對轉譯的調控

Abstract

EB 病毒 (Epstein-Barr virus, EBV) 又稱為第四型人類皰疹病毒。EB 病毒的生活史可分為潛伏期 (latency) 以及溶裂期 (lytic cycle)。在溶裂期極早期時會表現轉錄因子 Rta 蛋白質，活化溶裂期早期基因與晚期基因的表現，促使病毒顆粒的合成與組裝，並普遍認為不在病毒的潛伏期時表現；本實驗室先前的研究發現表現 Rta 會與 18S rRNA 及 mRNA 5’端帽結合且與核糖體有所關連，並可作為 ITAF 幫助下游基因 BZLF1 的轉譯，因此推測 Rta 可能參與調控細胞的轉譯作用。本研究發現過量表現 Rta 會使 HEK293T 細胞中 eIF4G 表現量下降。利用慢病毒建立了 Rta 靜默化的細胞株，結果發現潛伏期 Rta 會抑制 eIF4E 及 eIF2 的磷酸化作用。另外，利用免疫共沈澱法證實 Rta 會與 eIF2 及核糖體小亞基組成蛋白 S6 結合。接著利用 puromycin 以及 L-azidohomoalanine (AHA) 標定新生蛋白質藉此監測 Rta 蛋白質對轉譯作用速率的影響，結果發現 Rta 能夠促進細胞內總體轉譯 (global translation) 的速率。此外，本實驗室先前研究中發現，在潛伏期時可透過西方墨點法觀察到 Rta 訊號，且可於共軛焦顯微鏡下觀察到 Rta 多會聚集於核仁中，本研究進一步以西方墨點法再次驗證在受到 EB 病毒潛伏感染 (latent infection) 的細胞中確實有的 Rta 表現，同時也以共軛焦顯微進觀察到潛伏期 Rta 多存在於細胞核中。 本研究依據上述結果提出假設，細胞核仁中的潛伏期 Rta 可以透過與 40S 核糖體結合隨之運送至細胞質，穩定 eIF4F 複合體及 43S 轉譯起始前複合體的結合，並抑制 eIF2 的磷酸化作用，進而促進宿主細胞的總體轉譯作用。

Epstein-Barr virus (EBV), is also known as human herpesvirus 4 (HHV4). The life cycle of this virus can be divided into two phases, latency and the lytic cycle. Rta, a transcription factor, is defined as an immediate-early protein of EBV, and is required for activating the viral lytic genes. In our previous study, we found Rta has been associated with ribosomes and 5’ cap structure of mRNA, and is involved in the translation of the downstream BZLF1 gene, acting as an ITAF (IRES trans-acting factor). Therefore, we speculate that Rta might participate in regulating global translation. This study found that overexpressing Rta reduced the levels of eIF4G in HEK293T cells. Meanwhile, latency Rta suppressed the phosphorylation of eIF2 and eIF4E in Rta-silencing B lymphocytes, which was generated by lentivirus infection. Moreover, co-immunoprecipitation assay demonstrated that Rta is associated with eIF2α and S6. In addition, this study monitored the global translation rate by puromycin-labeling and AHA-labeling assay, the results indicated that Rta enhances global translation in HEK293T.Besides, we unexpectedly observed weak staining of Rta in the nucleouus of latently infected B lymphocytes by immunoblotting and immunofluorescence in our previous study. In this study, Rta was detected by immunoblotting and immunofluorescence in P3HR1 cells, suggesting that Rta is expressed during latency. To sum up, this research proposes that latency Rta binds to 40S subunits in nucleolus, then being exported to the cytoplasm. Rta can stabilize the binding between eIF4F and 43S preinitiation complex, and suppress the phosphorylation of eIF2α, therefore promoting the global translation of host cell.