Virus-induced autophagy in the marine coccolithophorid Emiliania huxleyi
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摘要: 以海洋环境中具有重要生态功能的赫氏颗石藻（Emiliania huxleyi）BOF92及其特异性裂解病毒（E. huxleyi virus, EhV99B1）为研究对象，采用分子及细胞生物学方法探究海洋病毒感染诱导宿主细胞自噬特征及其调控机制。结果显示：病毒感染能诱导宿主细胞自噬，并出现明显的液泡酸化及液泡自噬现象；感染过程中核心自噬相关基因如atg1、atg5、atg8及atg12的mRNA表达水平均显著上调（p < 0.01），自噬启动成核相关蛋白Vps34显著上调（p < 0.01），进而启动自噬并促使自噬体与液泡的融合；自噬标志性蛋白p62显著下调（p < 0.05），表明自噬流畅通以加速蛋白降解；感染中后期对自噬起负调控作用的磷脂酰肌醇（PI3K）、磷酸化蛋白激酶B（p-Akt）和磷酸化雷帕霉素靶蛋白（p-TOR）等因子的表达水平均显著降低（p < 0.01）。另外，病毒感染过程中，细胞活性氧（ROS）水平显著升高（p < 0.01），线粒体膜电位（MMP）及ATP含量显著降低（p < 0.01）。综上，病毒感染诱发宿主藻细胞ROS的产生、线粒体膜受损，并通过调节PI3K/Akt/TOR级联反应诱导细胞自噬。可见，自噬作为一种独特的程序性细胞死亡形式，对浮游植物遭受胁迫后的个体存活及种群延续具有重要意义。Abstract: To understand the characteristics of autophagy induced by virus infection in microalgae Emiliania huxleyi, we used diverse techniques including transmission electron microscopy, fluorescence microscopy, immunolabeling and biochemical methodologies to investigate the role of autophagy in the interaction between E. huxleyi BOF92 and its specific virus EhV99B1. The results showed that virus infection induced autophagy and vacuolar acidification in host cells, concomitant with up-regulation of autophagy-related genes such as atg1, atg5, atg8 and atg12 (p < 0.01) and Vps34 protein involved in the induction and nucleation of autophagosomes (p < 0.01). The expression level of autophagy marker protein p62 was significantly down-regulated (p < 0.05) during viral infection, indicating enhanced autophagic flux and activated autophagy. The expressions levels of negative regulatory factors such as phosphatidylinositol (PI3K), phosphorylated protein kinase B (p-Akt) and phosphorylated target of rapamycin protein (p-TOR) were significantly decreased in the late stage of viral infection (p < 0.01). Moreover, the level of reactive oxygen species (ROS) increased dramatically (p < 0.01), accompanied by a significant reduction in mitochondrial membrane potential (MMP) and ATP levels (p < 0.01) during viral infection. In conclusion, EhV99B1 infection induces ROS production and mitochondrial membrane damage in host cells, and initiates autophagy by regulating the PI3K/Akt/TOR signal pathway. Therefore, autophagy, as a unique form of programmed cell death, is of great significance to the individual survival and population dynamics of phytoplankton respond to environmental and biological stress.
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Key words:
- Emiliania huexlyi /
- virus /
- autophagy /
- autophagy-related genes /
- mitochondrial function /
- PI3K/Akt/TOR signal pathway
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图 1 EhV99B1感染诱导颗石藻（E. huxleyi） BOF92细胞的裂解及细胞超微结构变化
a−c. 病毒感染导致藻细胞裂解（a. 未添加病毒的对照组；b. 病毒感染24 h；c. 病毒感染48 h）；d−i. 病毒感染过程中藻细胞超微结构的变化（d. 对照组；e−g. 病毒感染24 h；h−i. 病毒感染48 h）；N表示细胞核；C表示叶绿体；M表示线粒体；V表示液泡；黑色箭头指向自噬体或病毒颗粒，图e−h右下角的方框为该图中箭头所指位置的局部放大图
Fig. 1 Infection dynamics and ultrastructure changes of E. huxleyi BOF92 during EhV99B1 infection
a−c. Virus infection leads to algae cell lysis (a. uninfected culture; b. 24 h post infection; c. 48 h post infection); d−i. transmission electron micrograph of E. huxleyi cells during viral infection (d. uninfected cells; e−g. 24 h post infection; h−i. 48 h post infection); N: nucleus; C: chloroplast; M: mitochondrial; V: vacuole; black arrows point to autophagosome or virions, the insets in Fig. e−h show higher magnification of the boxed areas, depicting the double membrane vesicles
图 2 颗石藻细胞中酸化区室的Lysosensor荧光标记结果
对照组：未添加病毒；感染组：病毒感染48 h；雷帕霉素组：阳性对照，采用终浓度为10 μmol/L的雷帕霉素处理藻细胞48 h
Fig. 2 Results of Lysosensor fluorescent labeling of acidizing compartments in Emiliania huxleyi cells
Control group: uninfected cells; infected group: viral infected cells 48 h post infection; rapamycin group: 10 μmol/L rapamycin-treated cells for 48 h (positive control)
图 3 病毒感染对颗石藻细胞核心自噬相关基因表达水平的影响
a. 核心自噬相关基因qRT-PCR结果聚类热图（数值为感染组与对照组的比值），Con 6-1表示对照组1 6 h后基因表达水平，Inf 6-1表示感染组1 6 h后基因表达水平，以此类推；b. 自噬相关基因相对表达量；*表示差异显著（p < 0.05），**表示差异较显著（p < 0.01），***表示差异极显著（p < 0.001）
Fig. 3 Relative expression level effect of core autophagy-related genes in E. huxleyi cells during viral infection
a. qRT-PCR analysis profiles of autophagy related genes，the value is the ratio of infected group to control group. Con 6-1 represents the gene expression level of the control group 1 after 6 h, Inf 6-1 represents the gene expression level of the infected group 1 after 6 h, and so on; b. relative expression levels of selected autophagy related genes; * indicates a significant differ(p < 0.05), ** indicates more significant differ (p < 0.01)， *** indicates an extreme differ (p < 0.001)
图 4 病毒感染对颗石藻细胞核心自噬相关蛋白表达的影响
*表示差异显著（p < 0.05），**表示差异较显著（p < 0.01）；***表示差异极显著（p < 0.001）
Fig. 4 Expression of core autophagy-related proteins in E. huxleyi cells during viral infection
* Indicates a significant differ (p < 0.05), ** indicates more significant differ (p < 0.01)， ***indicates an extreme differ (p < 0.001)
图 5 病毒感染对颗石藻细胞ROS水平的影响
Rosup为阳性对照组；**表示差异较显著（p < 0.01）；***表示差异极显著（p < 0.001）
Fig. 5 Effects of virus infection on the levels of ROS in E. huxleyi cells
Rosup indicates positive control group; ** indicates more significant differ ( p < 0.01)， *** indicates an extreme differ ( p < 0.001)
图 6 病毒感染对颗石藻细胞膜电位MMP的影响
CCCP为阳性对照组；***表示差异极显著（p < 0.001）
Fig. 6 Effects of virus infection on the levels of MMP in E. huxleyi cells
CCCP indicates positive control group; *** indicates an extreme differ (p < 0.01)
图 7 病毒感染对颗石藻细胞中ATP含量的影响
**表示差异较显著（p < 0.01）
Fig. 7 Effects of virus infection on the contents of ATP in E. huxleyi cells
** indicates more significant (p < 0.01)
图 8 病毒感染诱导颗石藻细胞自噬示意图
图中粗箭头所示为atg1、atg5及atg12的mRNA表达水平、Atg8、PI3K、p-Akt及p-TOR的蛋白表达水平和ROS、MMP和ATP含量的变化
Fig. 8 Schematic diagram of virus induced autophagy in the E. huxleyi-EhV system
Thick arrows indicate the mRNA expression level of atg1, atg5, atg12, protein expression levels of Atg8, PI3K, p-Akt, p-TOR and contents of ATP, ROS, MMP
表 1 本研究自噬相关基因qRT-PCR引物序列
Tab. 1 Primer sequence of autophagy-related gene for qRT-PCR used in this study
基因
名称扩增片段
长度/bp引物
名称序列（5’→3’） atg1 106 F AGGGCAGCTTTGCGATTGT R TTGGCCTGTAGCTTCTGGTTG atg2 96 F CATGGGCGTGTTTGTCGAG R AAGGCGTAGAGGGAGGAGAAG atg4_1 118 F GCGTGCCTTTGGTTCACGTA R GTGCTTGCGCGAGTATCATCT atg3 146 F TCCTGGAGAAGGGTGTGTTGAC R CGTGACGAGGTACTGCTTGTTC atg5 117 F GAGCACTTCCTGCCTTTTGC R CGTCAGCAGGTCAAAGAGCA atg7 154 F GCAAACACACTCGAGGACTTCAA R TCTTGAGGTCTGCGAAGGTGA atg8_1 163 F GCCGCTGATTGACAAGAAGAA R CGTCCTTGTGGCTGTCGTAGA atg9 215 F GTCGCCCGTTTCGTCACTT R CGAGCCATCACAGCAGGTT atg10 181 F GGTGCTGGAGGAGCCAATCT R AGCGGCAGATGTGTATGCG atg12 149 F CGAACCGCTGCTCCTCTACT R GAAAGCACTGCGCCACATC atg13 249 F GCGAAACTGCGTCCAGAAGA R CGCTCGAGAAGCACGAGATG LST8 212 F TGGCAAACAACTTCTCGTGC R TCAAACTGGCCCATCGTGTA TOR_1 110 F CAGCACAATCTCCTTCTGCAC R TGCCGATTTGCGGGTAC TOR_3 79 F GACCGGCACCTCAACAACA R CCTCGAAGCAGTCGCCATA Vps15 122 F CATCAAGGGCGAGAATGTGC R CGTCAAAGTAGAAGGAGAAGTCCG Vps34_2 203 F AGCTCGTCTGGAAGTTCCGGTA R CCTCGGCTGTATCTCGCATACAT β-tubulin 160 F TCATGTGCTCCTACTCGGTCTTC R TTCAGCGTGCGGAAACAGA 
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表 2 本研究中使用的抗体
Tab. 2 Antibodies used in this study
抗体名称 生产商 产品货号 稀释倍数 CDK1 Beyotime, 中国 AF1516 1∶800 ATG8 Abcam, 英国 ab4753 1∶1000 Vps34 Abcam, 英国 ab233437 1∶200 CDK1 Beyotime, 中国 AF0111 1∶1000 PI3K Beyotime, 中国 AF7749 1∶1000 p-AKT (Thr308) Beyotime, 中国 AF5734 1∶1000 p-TOR (Ser2448) Bioworld, 中国 BS4706 1∶1000 IgG Thermo, 美国 31460 1∶10000
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