絲氨酸235是C型肝炎病毒非結構性蛋白質5A的主要磷酸化位點可增進其與內質網膜的結合與病毒複製體形成

Abstract

C型肝炎病毒 (hepatitis C virus , HCV) 非結性構蛋白質5A (non-structure protein 5A , NS5A) 在病毒生活史中扮演重要的角色。許多研究以將NS5A上絲氨酸 (serine, S) 突變的方式，發現了一些可能參與NS5A的高度磷酸化態且對病毒生活史重要的磷酸化位點。然而，對於NS5A高度磷酸化態是否存在著不同的種類，與高度磷酸化態中不同的磷酸化位點彼此是否有互相依存的關係是尚未被研究過的。利用可以專一辨認磷酸化位點的抗體，我們分析了高度磷酸化的NS5A中S232, S235與S238的磷酸化情形。發現NS5A存在著不同的高度磷酸化態。分別約有6%, 92%與46%的高度磷酸化態NS5A具有S232, S235與S238的磷酸化。其中S232的磷酸化不須依賴S235或S238的磷酸化即能發生，但S238的磷酸化卻依賴S235的磷酸化才能發生。此結果暗示著S232, S235與S238的磷酸化是以S232為起點按順序、由casein kinase 1 這個具有辨認-3位置胺基酸是否磷酸化而決定磷酸化進行與否的激酶所介導完成的。S238的磷酸化可能僅是此一連續磷酸化下的副產物，對病毒生活史進行並無貢獻。而S235的磷酸化是最主要且具功能的磷酸化位點，S235磷酸化發生的時間與地點和病毒複製的情形相符。且在機制上，我們發現了S235磷酸化的NS5A會促進病毒複製體 (replication complex) 聚集於內質網膜狀結構中的脂筏 (lipid raft)上，有利於HCV複製的進行。

Phosphorylation of the hepatitis C virus (HCV) non-structure protein 5A (NS5A) plays critical roles in the viral life cycle. Based on genetic mutations followed by functional assays, numerous studies have pinpointed several serine residues that are critical for NS5A hyper-phosphorylation and for the viral life cycle. However, the hyper-phosphorylated NS5A species were not directly measured and whether phosphorylation among these sites is interdependent was unknown. Using three phosphorylation-specific antibodies, we profiled the NS5A species with single, double or triple phosphorylation at S232, S235 and/or S238. S232, S235 and S238 constitute respectively about 6, 92 and 46% of the hyper-phosphorylated NS5A. S232 phosphorylation occurs independently of S235 and S238 phosphorylation whereas S238 phosphorylation occurs only when S235 is phosphorylated. Our data suggest that S232 phosphorylation primes S235 by casein kinase 1  that requires priming phosphorylation at the -3 amino acid. S238 phosphorylation is perhaps a by-stander event dispensable for viral activity. S235 phosphorylation is the major and functional phosphorylation event that occurs at the time and intracellular location corresponding to viral replication. Mechanistically, S235-phosphoryalted NS5A probably facilitates clustering of the replication protein complexes in the ER lipid raft membranes for HCV replication.