B型肝炎病毒核心蛋白與壓力顆粒形成和先天性免疫反應之關聯性

Abstract

先天性免疫系統對於啟動適應性免疫反應以及病毒的清除至關重要。然而，B型肝炎病毒（Hepatitis B virus; HBV）長久以來一直被認為不能夠誘導有效的先天性免疫反應，因此被認為是一種「隱形病毒」。在被B型肝炎病毒感染的成年患者之中，大部分患者體內都能夠檢測到適應性免疫反應，並能有效地在6個月內將病毒清除，這表示作為連接適應性免疫反應橋樑的先天性免疫反應仍應有被病毒感染所誘導。近年來，越來越多病毒被證明能夠在宿主體內誘發壓力顆粒（Stress granule）的形成，許多病毒蛋白或核酸也被發現會與壓力顆粒中的分子交互作用，而壓力顆粒的產生與某些病毒誘導的先天性免疫反應具有一定關係。雖然目前有一些間接證據顯示B型肝炎病毒可能具有誘發壓力顆粒產生的能力，但對於B型肝炎病毒與壓力顆粒之間相關性的研究仍較為缺乏，因此本研究的目的在於探討B型肝炎病毒與壓力顆粒形成以及先天性免疫反應之間的關聯性，我們假設在B型肝炎病毒存在的情況下，宿主細胞會形成壓力顆粒並介導B型肝炎病毒誘發的先天性免疫反應，我們亦假設具有高度免疫原性的HBV核心蛋白（HBV core protein）會參與在HBV誘發壓力顆粒生成的機制當中。 在本研究中，我們利用可穩定表達HBV genotype D的HepAD38細胞株以及轉染 HBV genotype A質體的人類肝炎細胞株Huh7作為我們實驗的細胞株；同時也利用對細胞中壓力顆粒的標記蛋白G3BP1進行染色來確立壓力顆粒的生成。我們首先透過施加不同濃度以及時間的偏亞砷酸鈉（Sodium arsenite; NaAsO2）給予細胞氧化壓力，以建立可於細胞中穩定形成壓力顆粒的positive control，並篩選了適合對細胞中HBV抗原進行染色的抗體。接著我們觀察了在不同誘導天數的HepAD38中壓力顆粒的形成情形。我們觀察到在大多數細胞中，壓力顆粒並不會隨著HBV誘導天數的增加而產生，我們僅在誘導後第4、6、8天的少量細胞中觀察到些許壓力顆粒的產生，此外我們欲透過在誘導HBV產生的HepAD38細胞中添加偏亞砷酸鈉來觀察HBV核心蛋白以及壓力顆粒的共定位。然而，我們所使用的S162NP rabbit anti-HBc抗體並無法在誘導的天數中有效地對HBV核心蛋白進行染色，因此無法觀察到其在細胞中的分布及定位；有趣的是，利用偏亞砷酸鈉所誘導的壓力顆粒並沒有因為HBV的產生而受到影響，這可能表明genotype D的HBV無法穩定地誘導細胞中壓力顆粒的產生，並且也不會抑制由氧化壓力所造成的壓力顆粒形成。我們接下來透過轉染pAAV/HBV1.2 genotye A質體後不同天數的Huh7觀察不同基因型的HBV是否在誘導壓力顆粒上具有差異，結果顯示不論是在轉染pAAV質體或是在轉染pAAV/HBV1.2 genotype A質體的Huh7細胞中皆沒有觀察到壓力顆粒的產生，表明Genotype A的HBV在轉染的模式上並不會誘導壓力顆粒的生成。最後我們假設HBV可能含有潛在的機制去抑制壓力顆粒形成，因此我們透過轉染帶有不同突變的pAAV/HBV1.2 genotye A質體來觀察特定的HBV蛋白（HBc、HBe和HBx）的缺失和結構改變或是spliced RNAs的缺失是否會誘導壓力顆粒的生成，或是利用給予細胞core protein allosteric modulators（CpAM）檢視nucleocapsid的完整性對於壓力顆粒的形成是否具有影響，結果顯示特定HBV蛋白及spliced RNAs或是nucleocapsid的完整性皆與壓力顆粒的抑制或形成無關聯。總結以上，我們發現HBV可能無法於細胞模式中普遍地誘導壓力顆粒，但特定HBV蛋白（HBc、HBe和HBx）或是spliced RNAs也不具有抑制細胞中的壓力顆粒生成的機制，表示HBV所誘導的免疫反應可能是獨立於壓力顆粒的，因此HBV與先天性免疫反應之間的關係仍須要進一步探討及釐清。

Innate immune system are crucial for initiating adaptive immune responses and the subsequent clearance of viruses. Hepatitis B virus (HBV) has long been considered an "invisible virus" as it is thought to be incapable of inducing effective innate immune responses. However, most adult patients infected with HBV exhibit adaptive immune responses that effectively clear the virus within six months, suggesting that the innate immune response, which bridges to adaptive immunity, must still be induced during HBV infection. Recent studies have shown that many viruses can induce the formation of stress granules in host cells, with various viral proteins or nucleic acids interacting with molecules within these membraneless organelles. The formation of stress granules is associated with certain virus-induced innate immune responses. Although some indirect evidence suggests that HBV may induce stress granule formation, research on the relationship between HBV and stress granules remains limited. Therefore, the aim of this study is to investigate the association between HBV, stress granule formation, and innate immune responses. We hypothesize that in the presence of HBV, host cells will form stress granules that mediate HBV-induced innate immune responses. We also propose that the highly immunogenic HBV core protein involves in the mechanism of stress granule formation. In this study, we used the HepAD38 cell line, which stably expresses HBV genotype D, and the Huh7 human hepatoma cell line transfected with HBV genotype A plasmids as our experimental cell models. We also utilized staining for the stress granule marker protein G3BP1 to investigate stress granule formation. First, we applied sodium arsenite (NaAsO2) at various concentrations and durations to induce oxidative stress in cells, establishing a positive control for stable stress granule formation. We then screened for antibodies suitable for staining HBV antigens within the cells. Subsequently, we observed the formation of stress granules in HepAD38 cells at various induction days. We found that stress granules did not form with increasing HBV induction days in most cells; only a few cells showed stress granule formation on days 4, 6, and 8 post-induction. Additionally, we aimed to observe the co-localization of the HBV core protein and stress granules by adding sodium arsenite to HepAD38 cells producing HBV. However, the S162NP antibody we used did not effectively stain the HBV core protein during the induction days, making it impossible to observe its distribution and localization within the cells. Interestingly, the stress granules induced by sodium arsenite were not affected by the presence of HBV, suggesting that HBV genotype D does not stably induce stress granule formation and does not inhibit stress granules formed by oxidative stress. Next, we observed whether different HBV genotypes differ in inducing stress granules by transfecting Huh7 cells with the pAAV/HBV1.2 genotype A plasmid. The results showed that stress granules did not form in Huh7 cells transfected with either pAAV vectors or pAAV/HBV1.2 genotype A plasmids, indicating that genotype A HBV does not induce stress granule formation in the transfection model. Finally, we hypothesized that HBV might have mechanisms to inhibit stress granule formation. To test this, we transfected cells with pAAV/HBV1.2 genotype A plasmids containing various mutations to examine whether the deletion or structural alteration of specific HBV proteins (HBc, HBe, and HBx) or the absence of spliced RNAs would induce stress granule formation. Additionally, we assessed the impact of nucleocapsid integrity on stress granule formation by treating the cells with core protein allosteric modulators (CpAM). Our results indicated that neither specific HBV proteins, spliced RNAs, nor nucleocapsid integrity were associated with the suppression or formation of stress granules. In summary, we found that HBV may not generally induce stress granules in cellular models. However, specific HBV proteins (HBc, HBe, and HBx) or spliced RNAs also do not appear to possess mechanisms that inhibit the formation of stress granules in cells. This suggests that the immune response induced by HBV may be independent of stress granules, indicating that the relationship between HBV and the innate immune response requires further investigation and clarification.