HBV 核殼蛋白的 C 端區域在 HBV 先天免疫反應中所扮演的角色

Abstract

病原體相關小分子 (PAMPs) 主要分成核酸及蛋白質兩類，被專一性受體 (PRRs) 辨認後，會促進干擾素 (interferon) 產生，進而引發後天免疫反應，產生毒殺性 T 細胞 (cytotoxic T cell) 及抗體。根據文獻指出，在利用尾靜脈注射 (hydrodynamic tail vein injection) 包含 HBV genome 的質體，模擬 HBV 感染的小鼠模式中，HBV 被清除的情況並非依靠產生干擾素 (interferon) ，並不會因常見的辨認病毒核酸的 PRRs 被剔除而有所改變。HBV的先天性免疫並非依靠產生干擾素 (interferon)，以及促進干擾素刺激基因(interferon-stimulated genes)表現，卻依然能活化後天免疫反應，達到病毒清除的目標。另外根據陳培哲教授團隊先前的研究指出，組成 HBV 核殼 (capsid) 的核殼蛋白 (core protein) 可能對於清除 HBV扮演重要角色，其必須保有最後 10 個氨基酸且在 capsid form 的立體結構中，才有可能作為 PAMP 被 PRR 辨認。本論文目標為進一步找出 HBV 的核殼蛋白最後 10 個氨基酸相關的 PAMP 為何，及其如何引發先天及後天免疫反應清除HBV。由於實驗室先前研究發現核殼蛋白C端最後一個第 183 個氨基酸 Cysteine (183Cys) 會參與形成由雙硫鍵鍵結形成的核殼蛋白 dimer 結構，且 HBV 核殼可以由具有 183 個氨基酸和 182 個氨基酸的核蛋白組成，因此本研究提出一假說，即HBc183形成的雙硫鍵結，於capsid中有可能成為PRR所辨認之特殊結構，影響先天免疫反應。為測試此假說，將利用HDI mouse model，經由尾靜脈注射具有細胞內複製能力的HBV質體，探討HBc與HBc-Cys在HBV引發的免疫反應中是否扮演的不同角色。使用尾靜脈注射有不同核蛋白表現的 AAV-HBV replicon 質體，分別為形成只有 183 個氨基酸、只有 182 個氨基酸、第 183 個氨基酸由 Cysteine 突變成 Alanine 的核蛋白組成的核殼，於每週追蹤血清中 HBV 表面抗原的變化，以代表 HBV 清除效率。結果發現 wild-type 及只有 183 個氨基酸組別HBV清除效率較只有 182 個氨基酸之組別快速。此結果指出HBc183形成的雙硫鍵結之特殊結構，於capsid中的確可能影響免疫反應，值得未來利用此動物模式進一步探討其分子機制。

The pathogen-associated molecular patterns (PAMP), either nucleic acid or protein, recognized by the cellular pattern recognition receptors (PRR) is critical for inducing the immune responses for clearance of pathogens. In most cases, recognition of PAMP by PRR promotes interferon production for activation of subsequent adaptive immune responses. As documented, HBV infection does not induce interferon production but still can activate the adaptive immune responses for viral clearance. Identification of the putative PAMP and PRR is still remained an unsolved issue for HBV. Using the tail vein hydrodynamic injection mouse model, Prof. Pei-Jer Chen’s group has previously identified the critical role of HBV core protein (HBc) in inducing the immune responses, mainly dependent on the intact capsid structure and the 10 amino acids at the C-terminal of HBc. However, the exact structure and underlying mechanism for the C-terminal domain of HBc is not clear yet. In our previous study that the very C terminal cysteine of HBc could contribute to the formation of disulfide bond in capsid, which however is lack in the HBc-Cys protein synthesized by the splicing SP1 RNA. Regarding that both HBc and HBc-Cys is contained in the viral capsid, it will be interesting to test if the disulfide bond structure mediated by the C terminal cysteine of HBc could be the structure responsible for inducing the immune responses. We propose to test this hypothesis by the tail vein hydrodynamic injection mouse model, by injection of the wild type and mutant HBV replicon constructs only expressing either HBc or HBc-Cys protein only and follow up of the serum HBsAg and anti-HBs Ab levels. The results showed a similar clearance rate between the mice injected with wild type and HBc-only replicon constructs, which is intriguingly much faster than the mice injected with HBc-Cys-only replicon construct. The results indicated that the C terminal cysteine of HBc could contribute to the immune clearance of HBV and deserves further dissection of the underlying mechanism in this mouse model.