冠狀病毒磷酸化核殼蛋白參與調控β型干擾素生成之角色研究

Abstract

無法有效誘導先天性免疫反應為冠狀病毒感染造成其嚴重致病的原因之一，多數冠狀病毒感染時會使干擾素生成受到抑制。許多病毒蛋白被認為參與在拮抗先天性免疫反應中，其中包括核殼蛋白，但詳細機制目前尚未明瞭。本研究的目的為探討冠狀病毒躲避干擾素生成的機制。利用老鼠冠狀病毒原型：鼠肝炎病毒 (mouse hepatitis virus, MHV) 的JHMV strain作為研究材料，我們發現當給予干擾素處理，病毒RNA生成和病毒力價 (viral titer)會因此降低；而如同預期，JHMV感染無法誘導干擾素生成與活化下游抗病毒之訊息傳遞路徑。在我們先前的研究中指出，DDX1與磷酸化核殼蛋白的交互作用參與調控JHMV的不連續轉錄機制進行，而DDX1為一RNA解旋酶，過去研究中曾報導其能夠作為sensor進而誘導干擾素產生，本研究因此提出探討在JHMV感染時，DDX1與磷酸化核殼蛋白的複合體參與調控先天興免疫反應的機制。 在穩定表現JHMV核殼蛋白的細胞中過量表現DDX1-Myc或是enzyme dead DDX1-Myc (K52A) 能夠誘導β型干擾素 (interferon beta, IFN-β) 產生，當給予GSK-3抑制劑抑制核殼蛋白磷酸化時，IFN-β生成則受到抑制，此結果顯示DDX1透過和磷酸化核殼蛋白的交互作用誘導的IFN-β生成，且與DDX1酵素活性無關。為了進一步了解其誘導機制，我們發現當knockdown RIG-I或是MAVS以及施予TBK1抑制劑時，能夠顯著減少IFN-β生成，此處理同時會造成DDX1表現量增加。根據此結果可推論DDX1與磷酸化核殼蛋白是透過活化RIG-I/MAVS/TBK1途徑進而誘導IFN-β生成，然而透過此途徑會進一步降低DDX1表現量造成IFN-β生成減少，可能為核殼蛋白參與拮抗先天性免疫反應之一可能機制。然而RIG-I/MAVS/TBK1途徑與DDX1/磷酸化核殼蛋白的新穎回饋循環 (feedback loop)調控機制仍有待進一步探討。

關鍵字：冠狀病毒、先天性免疫反應、DDX1、干擾素、核殼蛋白。

One major pathogenic mechanism for severe coronavirus (CoV) infection is the failure to induce effective innate immune responses. The interferon (IFN) production is suppressed in most CoV infections. Several viral proteins have been identified involved in antagonizing the innate immune response, including the nucleocapsid (N) protein, but the mechanism still remained unclear. This study aims to study the mechanism for CoV to escape the IFN induction. Using the JHMV strain of mouse hepatitis virus (MHV), the prototype of CoV, we first found that the viral RNA production and viral titers were decreased upon IFNtreatment, and as expected, JHMV infection cannot induce IFN production and activate downstream antiviral signaling pathways. In our previous study, an interaction between DDX1 and phosphorylated N protein (pN) was identified involved in regulating the discontinuous transcription in JHMV. As DDX1, an RNA helicase, has been reported to function as a sensor to induce IFN responses, this study proposed to examine if DDX1 in complex with the pN protein is involved in regulating the IFN innate immune responses during JHMV infection. Overexpression of either DDX1-Myc or the enzyme dead DDX1-Myc(K52A) in JHMV-N protein stably expressed DBT cells induced IFN-β production. Such an induction was abolished by treatment with the GSK-3 inhibitors, which significantly decreases the phosphorylation of N. The results suggested that DDX1 could via interacting with pN to induce IFN production, in an enzyme independent manner. To further dissect the mechanism, we found that knockdown of either RIG-I or MAVS expression and the treatment of TBK1 inhibitor significantly decreased the production of IFN-β, and interestingly the DDX1 was elevated at the same time. The results suggested that DDX1 and JHMV-pN induces IFN-β production through activation of the RIG-I/MAVS/TBK1 axis. This axis however in turn decreases the DDX1 level and thus leads to a decrease of IFN-β production. The detailed mechanism underlying this novel feed-back regulatory loop between RIG-I/MAVS/TBK1 axis and DDX1/CoV-pN awaits further investigation.

Key words: coronavirus, innate immunity, DDX1, interferon, nucleocapsid.