病毒介导的海洋球石藻新型鞘脂类代谢调控机制及其诱导宿主细胞凋亡研究

Coccolithophores are unicellular marine eukaryotic microalgae. Emiliania huxleyi is the host for the coccolithovirus (EhV), which is responsible for the demise of large oceanic blooms formed by this alga. Marine viruses that infect phytoplankton are recognized as a major ecological and evolutionary driving force, shaping community structure and nutrient cycling in the marine environment. The EhV-86 virus genome sequence has identified several genes apparently involved in sphingolipid metabolism. The EhV-86-encoded genes contain only a subset of the activities required to generate the novel sphingolipid, implying that its synthesis is the result of coordinated interactions between algal-and viral-encoded biosynthetic enzymes. The recent availability of genomic resources for an E. huxleyi host CCMP1516 and E. huxleyi lytic virus strain EhV86 provides an unprecedented opportunity to explore cellular pathways triggered during execution of viral infection and to gain insights into the origin of apoptosis in E .huxleyi. .Currently, very little is known about the molecular mechanisms mediating E. huxleyi death by marine viruses. So it will be important to identify the function of genes that encodes enzymes involved in host sphingolipid metabolism, particularly given the proposed role for this sphingolipid in apoptosis. Gene silencing can occur either through repression of transcription, or through mRNA degradation. Because of its high specificity and efficiency, RNA interference has been proven to be an invaluable tool for analyzing the biological function of the target gene and adjusting the metabolism process considerably by sequence-specific knockdown. .In this subject, we combined genetic engineering and RNAi technology to explore the cellular pathways mediating the interaction between E. huxleyi BOF92 and its specific coccolithoviruses EhV99B1 strains. We examined the sphingolipid biosynthetic pathway by enhancing the expression of EhV-SPT gene encoding the first and committed enzyme in de novo sphingolipid synthesis, and reducing the expression of EhV-GCS gene encoding the key enzyme in ceramide inverting to glucosylceramide and then to gain a better understanding of the molecular mechanisms underlying the E. huxleyi–virus interaction.

海洋球石藻是一种全球广泛分布且具有重要生态功能的真核浮游植物。自然海域中，病毒感染和裂解是终止该藻赤潮调控其种群丰度的主要因素之一。一株海洋球石藻及其病毒全基因组注释发现，病毒通过基因横向转移从宿主基因组中获得了一些列鞘脂类代谢关键酶的基因 ，并可能在一定程度上掌控了宿主鞘脂类代谢，大量合成、积累病毒性鞘脂类物质并诱导宿主细胞凋亡。这些酶在宿主鞘脂类代谢途径中的确切位置及功能目前尚不明确。本研究拟以海洋球石藻及其特异性病毒为研究对象，利用RNAi、基因工程及分子细胞生物学等技术，选择性过表达或干扰病毒基因组中鞘脂类代谢的关键酶基因，探讨基因表达水平的改变对宿主鞘脂类代谢产物的形成、积累及其诱导宿主细胞凋亡的影响，确定这些关键酶在宿主鞘脂类代谢途径中所处的环节及功能。初步揭示病毒介导的球石藻宿主新型鞘脂类代谢的分子调控机制，为深入了解海洋球石藻病毒与宿主间复杂的相互作用关系提供新的理论认识。

本研究阐释了海洋球石藻病毒调控宿主鞘脂代谢及诱导细胞凋亡的分子机制。①通过基因过表达及鞘脂组学分析，确定了病毒3个鞘脂代谢相关基因调控宿主鞘脂代谢的功能：EhV-SPT（丝氨酸棕榈酰转移酶），EhV-FAD（脂肪酸去饱和酶）和EhV-SD（甾醇去饱和酶）均能恢复相应基因缺陷型酵母相关脂质的合成。SPT利用鞘脂的从头合成途径并偏好以C15-CoA（肉豆蔻酰辅酶A）而非常规的C16-CoA（棕榈酰辅酶A）为底物，合成基于奇数C17-LCB长链的病毒性鞘脂；FAD通过影响酵母脂肪酸代谢（诱导脂肪酸由多不饱和向单不饱和代谢漂移）间接调控鞘脂代谢；SD则主要通过催化酵母甾醇等衍生脂类的代谢从而影响鞘脂代谢。②病毒感染重构宿主鞘脂代谢，其组成和含量呈现出进展性（progressive）和非进展性（non-progressive）变化特点，显著变化的鞘脂Cer 38:1;2和Cer 40:2;2筛选为响应病毒感染的鞘脂标志物；感染后期病毒性鞘脂大量合成，作为病毒粒子的结构成分贡献于病毒的组装和释放并作为信号分子诱导宿主细胞凋亡。③病毒性鞘脂诱导的细胞凋亡涉及metacaspase的磷酸化活性形式，并可能与磷脂酶C信号通路相关。E. huxleyi基因组编码了核心自噬相关蛋白的关键功能单位，病毒感染通过调控自噬及凋亡相关基因在时空上的差异表达逐步诱导细胞死亡，且病毒诱导的自噬可能受控于其自身转录的非编码tRFs（transfer RNA related fragments）的调控。④从病毒感染的球石藻细胞中纯化出一种新型鞘脂类单体化合物（C40H79NO3），在20g/mL浓度下对肝癌细胞Hep G2的抑制率达到62.64%，有望开发为抗肝癌新药。.本项目基于鞘脂代谢证实了海洋真核藻类病毒不仅是宿主代谢产物的被动消费者，还可以象噬菌体一样通过编码自身的辅助代谢基因重构宿主代谢网络来扩大宿主的代谢能力，以支持病毒的特殊需求，使我们对“病毒细胞代谢（virocell metabolism）”这一独特的新兴代谢模式在海洋病毒-宿主激烈的“军备竞赛”过程中既对抗又协同进化的生态学意义有了新的、更深层次的认知。另外，EhV诱导的细胞死亡可能存在一个全新的转录后水平的基因表达调控，为探索浮游植物病原菌胁迫调控开辟了新思路。