SARS-CoV-2 非結構性蛋白質1 (NSP1) 調控 RIG-I/MDA-5 訊息傳遞之角色

Abstract

嚴重急性呼吸道症候群冠狀病毒2型 (Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)於2019年底首次於中國大陸武漢省發現，經基因體定序確認後為新型冠狀病毒，屬於Coronaviridae中的Betacoronavirus，感染後主要造成咳嗽和發燒較嚴重的症狀包括肺炎和腎衰竭甚至死亡，截至2023年底共造成7.7億人口感染並導致約700萬人死亡，死亡率約為0.9%。先前研究報告指出SARS-CoV-2非結構性蛋白質1 (non-structural protein 1，NSP1)會抑制第一型和第三型干擾素的啟動子活性。在本研究中，藉由冷光素酶活性分析發現NSP1具有抑制冷光素酶活性的作用，但進一步分析發現其抑制作用並不是直接影響啟動子的活性而是透過抑制冷光素酶的表現。在發現NSP1具有抑制蛋白質轉譯的功能後，本研究針對參與RIG-I/MDA-5訊息傳遞路徑的蛋白質是否會受到NSP1的影響進行探討，結果發現NSP1除了會抑制MDA-5和IFIT3的mRNA和蛋白質表現，同時也會抑制IRF3磷酸化，但卻不影響IFN-β和IFN-λ mRNA表現量。另一方面，實驗結果也發現不具有抑制轉譯功能的點突變NSP1蛋白質，NSP1(K164A/H165A)不會抑制MDA-5和IFIT3的蛋白質表現量，推測NSP1利用抑制轉譯的方式來影響干擾素訊息傳遞路徑相關蛋白質如: MDA-5和IFIT3的表現來抑制干擾素訊息傳遞。

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared in December 2019 in Wuhan, China. After virus genome sequencing and identification, the virus was recognized as a novel coronavirus, which became the 7th coronavirus that can infect human. SARS-CoV-2 belongs to Betacoronavirus, one of the four genera of Coronaviridae. SARS-CoV-2 infection causes cough, fever, and sometimes pneumonia and renal failure or even death. Until December 2023, SARS-CoV-2 has infected 770 million people and caused 7 million deaths, the mortality rate is about 0.9%. In previous studies, SARS-CoV-2 non-structural protein 1 (NSP1) was demonstrated having ability to inhibit type I and type III interferon (IFN) promoter activity. In this study, luciferase reporter assay also showed inhibitory effects of NSP1 on IFN-β and IFN-λ promoter- driven luciferase activity. Nevertheless, the reduced luciferase activity was at least partially, due to an inhibition on luciferase expression rather than a direct effect on the promoter. Whether NSP1 has effects on the expression of protein involving in RIG-I/MDA-5 signaling pathway was then investigated. The results showed that both mRNA and protein expression of MDA-5 and IFIT3 were inhibited by NSP1, and the phosphorylation of IRF3 was also inhibited by NSP1. However, these inhibitory effects did not reflect on the transcription of IFN-β and IFN-λ since the IFN-β and IFN-λ mRNA levels were not affected. On the other hand, NSP1(K164A/H165A), which has lost the function of translation inhibition, had no effect on MDA-5 and IFIT3 protein expression, indicating that NSP1 reduces the protein level of MDA-5 and IFIT3 mainly through translation inhibition. Taken together, this study demonstrated that NSP1 has ability to inhibit expression of specific proteins that participate in interferon signaling.