SARS-CoV類病毒顆粒感染巨噬細胞對NFkB路徑之調節作用

Abstract

2003年春天，爆發一個新興感染症，其臨床表徵有38℃以上的高燒、乾咳以及肺部的淋巴球浸潤等，除此之外，發病後期雖然病毒量不高，但病患體內仍可偵測到大量的細胞激素 (cytokine)。後來將病人檢體接種到非洲綠猴腎臟細胞 (Vero E6)，成功地培養並分離出病原體，將它命名為嚴重急性呼吸道症候群冠狀病毒 (SARS-CoV)。病毒顆粒由四個主要的結構性蛋白質所組成，分別是棘蛋白質 (spike protein, S)、膜小蛋白質 (envelope protein, E)、膜蛋白質 (membrane protein, M) 和核套蛋白質 (nucleocapsid protein, N)。 過去mouse hepatitis virus和transmissible gastroenteritis virus冠狀病毒的研究顯示，同時表現冠狀病毒的E與M蛋白質，可以在培養液裡面收集到類病毒顆粒；而且transmissible gastroenteritis virus的E與M蛋白質所形成的類病毒顆粒還會刺激白血球產生甲型干擾素 (interferon a)。最近的研究顯示SARS-CoV刺激免疫細胞分泌細胞激素與病毒的複製並沒有直接的關係。 為瞭解SARS-CoV感染時對巨噬細胞產生的效應，本研究首先建立了SARS-CoV的類病毒顆粒系統。收集SARS-CoV E、M與S形成的類病毒顆粒，與老鼠巨噬細胞株RAW264.7共同培養時，發現細胞中IkBa的量有減少的現象，這種減少的現象可被proteasome inhibitor MG132所抑制，表示SARS-CoV類病毒顆粒活化了巨噬細胞的NFkB訊息傳遞路徑。若利用對抗SARS-CoV的抗體來中和類病毒顆粒，就觀察不到IkBa下降的現象，顯示IkBa的減少確實是由類病毒顆粒所造成的。目前已知SARS-CoV的S蛋白質與樹突細胞 (dendritic cell) 上的DC-SIGN有結合作用，因此本研究也進一步分析SARS-CoV類病毒顆粒負調控IkBa的現象與DC-SIGN的相關性。結果發現如果先以抗DC-SIGN的抗體處理細胞，則SARS-CoV類病毒顆粒造成IkBa下降的情形較不明顯，表示DC-SIGN可能參與在SARS-CoV活化NFkB的作用上。

SARS is an emerging disease discovered in 2003. The clinical symptoms included fever higher than 38℃, cough, and lymphocyte infiltration observed in chest X film. Besides, high level of cytokines could be detected even after the peak of viral load. Upon specimen inoculation, the causative agent was successfully cultured in VeroE6 cells and could be isolated from the culture medium. The causative agent was named SARS-CoV. SARS-CoV is composed of four major structure proteins, spike (S), membrane (M), envelope (E) and nucleocapsid protein (N). Previous studies with mouse hepatitis virus and transmissible gastroenteritis virus demonstrated that virus-like particles could be collected from culture medium when E and M proteins were co-expressed. Besides, VLPs composed of E and M proteins of transmissible gastroenteritis virus stimulated leukocytes to produce interferon α esults from a recent study indicated that cytokine stimulation in macrophage is independent of the replication ability of SARS-CoV. To understand the effects of SARS-CoV infection on macrophages, culture systems capable of producing SARS VLPs were established. When RAW264.7 macrophages were incubated with SARS-VLPs composed of S, M and E proteins, a reduction of IkBα level was detected. The reduction of IkBα could be blocked by proteasome inhibitor, MG132. These suggest that SARS VLPs stimulate the degradation of IkBα and activate NFkB. Mouse anti-60Co inactivated SARS-CoV antibodies neutralized the VLPs and eliminated the effect of SARS-VLPs on NFkB signaling pathway, further confirmed that the reduction of IkBα is indeed resulted from the presence of the SARS-VLPs. Recently, an interaction between SARS-CoV spike protein and DC-SIGN has been demonstrated. Therefore, a possible role of DC-SIGN involved in the reduction of IkBα was investigated. Preincubation of antibodies against DC-SIGN with RAW264.7 macrophages partially restored the negative effect of SARS VLPs on IkBα expression. This suggests that DC-SIGN may be involved in the NFkB activation caused by SARS-CoV.