EB 病毒 Rta 於 cGAS-STING 先天免疫反應中所扮演的角色

Abstract

EB 病毒 (Epstein-Barr Virus, EBV) 又稱為第四型人類皰疹病毒，全球約有 90% 以上的人受到感染，與多種癌症和自體免疫疾病相關。EB 病毒的生活史分為潛伏期 (latency) 與溶裂期 (lytic cycle)，甫進入溶裂期時會表現極早期蛋白質 Rta，主要作為轉錄因子調控早期與晚期基因表達，使病毒能複製基因體與製造 殼體蛋白質，進而組裝並釋放病毒顆粒。病毒感染時，宿主可以感應細胞質中的 外源病毒 DNA 或因壓力釋出的內源性胞器 DNA，透過 cGAS-STING 訊息傳遞 路徑產生第一型干擾素 (type I interferons)，以調節先天免疫反應。先前研究指出 Rta 會減少由 cGAS-STING 引起的第一型干擾素基因的轉錄。本研究先以 luciferase reporter assay 與定量反轉錄 PCR (RT-qPCR) 確認 Rta 能減少第一型干擾素基因轉錄以及所傳遞的下游免疫反應；並發現 Rta 的蛋白質量增加時，會減少細胞中過量表達的 STING 與免疫反應最下游的磷酸化 IRF3 蛋白質的表現量。接著以 cycloheximide (CHX) chase assay 確認 Rta 會透過增強 STING 蛋白質的細胞自噬 (autophagy)，降低 STING 的穩定性。本研究進一步以免疫共沈澱分析證實 Rta 會與 STING 蛋白質結合，並且發現 Rta 可以減少 STING 在內質網 (endoplasmic reticulum)上形成二聚體和多聚體。此外以變性蛋白免疫沈澱 (denaturing-immunoprecipitation) 分析，顯示 Rta 會干擾 STING 的 K63 鏈結的泛素化 (ubiquitination)，進而影響其功能調節。本研究證實 Rta 可以促進 STING 的細胞自噬降解以及干擾 STING 的泛素化，來抑制 cGAS-STING 訊息傳遞所引起的第一型干擾素先天免疫反應。

Epstein-Barr Virus (EBV), or human herpesvirus 4 (HHV4), infects 90% of the adult population worldwide and is associated with cancers, autoimmune diseases, and neurological disorders. The EBV life cycle is split into latency and the lytic phases; the immediate-early protein Rta expressed at lytic initiation transactivates the expression of EBV early and late genes, facilitating viral DNA replication, production of viral structural proteins, and virion assembly and egress. It is well known that viral DNA or DNA released from organelles triggers cGAS-STING signaling and ultimately activates the transcription of type I-interferon (IFN) genes to promote innate immunity. Previous research revealed that EBV Rta reduces the cGAS-STING-induced IFN-b promoter activities, although the underlying mechanism remains unclear. This study first confirmed that Rta decreased the transcription of the IFN-b gene and interferon-stimulated genes (ISGs). Rta also reduces the levels of STING and phosphorylated IRF3 in a dose-dependent manner, as Rta destabilizes STING by enhancing its autophagic degradation in a cycloheximide (CHX) chase assay. This study also shows the interaction between Rta and STING and that Rta reduces the formation of STING dimers and oligomers on the endoplasmic reticulum (ER) surface, which is critical to cGAS-STING signaling. Meanwhile, Rta perturbs total ubiquitination and K63-linked polyubiquitination on STING, possibly affecting STING functionalities. These findings illuminate Rta’s importance in regulating the cGAS-STING-mediated type I IFN response by promoting STING autophagic degradation and perturbing STING K63-linked polyubiquitination.