Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT
Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT
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DOI：
10.1101/2022.06.01.494398
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发表时间：
2022-07
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影响因子：
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通讯作者：
Tomasz H. Benedyk;V. Connor;Eve R. Caroe;M. Shamin;D. Svergun;J. Deane;C. Jeffries;C. Crump;S. C. Graham
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文献类型：
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作者：
Tomasz H. Benedyk;V. Connor;Eve R. Caroe;M. Shamin;D. Svergun;J. Deane;C. Jeffries;C. Crump;S. C. Graham
单纯疱疹病毒（HSV）-1在感染过程中显著改变细胞膜的结构和蛋白质组成，但其对膜脂质组成的影响尚不清楚。HSV-1 pUL 21是一种病毒编码的蛋白磷酸酶接头，可促进多种细胞和病毒蛋白（包括细胞神经酰胺转运蛋白CERT）的去磷酸化。CERT介导神经酰胺从ER到反式高尔基体网络的非囊泡转运，因此神经酰胺转化为鞘磷脂和在细胞增殖、细胞信号传导和膜运输中起重要作用的其他鞘脂。使用点击化学分析培养细胞中鞘脂代谢的动力学，我们表明pUL 21介导的去磷酸化激活CERT，并增加神经酰胺转化为鞘磷脂的速率。纯化的pUL 21和全长CERT以亚微摩尔亲和力相互作用，我们绘制了负责相互作用的结构域。使用小角X射线散射解决与CERT PH和START结构域复合的pUL 21 C-末端结构域的溶液结构，使我们能够鉴定pUL 21表面上破坏体外和培养细胞中CERT结合的单个氨基酸突变。鞘脂谱表明，在HSV- 1感染的情况下，神经酰胺向鞘磷脂的转化严重减少，当感染编码缺乏激活CERT能力的突变形式的pUL 21的病毒时，这种缺陷会加剧。然而，当pUL 21介导的CERT去磷酸化被消除时，病毒复制和传播没有显著改变，这突出了其他细胞和/或病毒靶标的去磷酸化支持了pUL 21在HSV-1生物学中的重要作用。单纯疱疹病毒（HSV）-1会导致神经元的终身休眠感染，偶尔会重新激活，表现为唇疱疹或生殖器疱疹。虽然HSV-1对感染细胞蛋白质含量的影响已经得到了很好的研究，但我们对它对细胞脂质的影响知之甚少。在培养细胞中使用生物正交标记，我们表明HSV-1蛋白pUL 21激活关键的细胞脂质转运蛋白CERT，以加速神经酰胺转化为鞘磷脂。HSV-1感染显著改变神经酰胺代谢的动力学，导致神经酰胺蓄积。防止CERT激活的HSV-1 pUL 21突变进一步增强了神经酰胺的积累，但这不会改变HSV-1的复制或传播，这凸显了其他细胞和/或病毒蛋白质代表了培养细胞中pUL 21介导的去磷酸化的关键靶点。
Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non- vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV- 1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread are not significantly altered when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. Significance Herpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of HSV-1 pUL21 to prevent CERT activation further enhances ceramide accumulation but this does not alter the replication or spread of HSV-1, highlighting that other cellular and/or viral proteins represent the critical targets of pUL21-mediated dephosphorylation in cultured cells.