Ott Melanie, Marques Débora, Funk Christina, Bailer Susanne M
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, 80336, München, Germany.
Institute for Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstrasse 12, 70569, Stuttgart, Germany.
Virol J. 2016 Oct 20;13(1):175. doi: 10.1186/s12985-016-0638-8.
Herpes simplex virus type 1 (HSV1), a member of the alphaherpesvirinae, can cause recurrent facial lesions and encephalitis. Two membrane envelopment processes, one at the inner nuclear membrane and a second at cytoplasmic membranes are crucial for a productive viral infection. Depending on the subfamily, herpesviruses encode more than 11 different transmembrane proteins including members of the tail-anchored protein family. HSV1 encodes three tail-anchored proteins pUL34, pUL56 and pUS9 characterized by a single hydrophobic region positioned at their C-terminal end that needs to be released from the ribosome prior to posttranslational membrane insertion. Asna1/TRC40 is an ATPase that targets tail-anchored proteins to the endoplasmic reticulum in a receptor-dependent manner. Cell biological data point to a critical and general role of Asna1/TRC40 in tail-anchored protein biogenesis. With this study, we aimed to determine the importance of the tail-anchored insertion machinery for HSV1 infection.
To determine protein-protein interactions, the yeast-two hybrid system was applied. Asna1/TRC40 was depleted using RNA interference. Transient transfection and virus infection experiments followed by indirect immunofluorescence analysis were applied to analyse the localization of viral proteins as well as the impact of Asna1/TRC40 depletion on virus infection.
All HSV1 tail-anchored proteins specifically bound to Asna1/TRC40 but independently localized to their target membranes. While non-essential for cell viability, Asna1/TRC40 is required for efficient HSV1 replication. We show that early events of the replication cycle like virion entry and overall viral gene expression were unaffected by depletion of Asna1/TRC40. Furthermore, equal amounts of infectious virions were formed and remained cell-associated. This indicated that both nuclear egress of capsids that requires the essential tail-anchored protein pUL34, and secondary envelopment to form infectious virions were successfully completed. Despite large part of the virus life cycle proceeding normally, viral propagation was more than 10 fold reduced. We show that depletion of Asna1/TRC40 specifically affected a step late in infection during release of infectious virions to the extracellular milieu.
Asna1/TRC40 is required at a late step of herpesviral infection for efficient release of mature virions to the extracellular milieu. This study reveals novel tools to decipher exocytosis of newly formed virions as well as hitherto unknown cellular targets for antiviral therapy.
单纯疱疹病毒1型（HSV1）是α疱疹病毒亚科的成员，可引起复发性面部病变和脑炎。两个膜包裹过程，一个在内核膜，另一个在细胞质膜，对于有效的病毒感染至关重要。根据亚科的不同，疱疹病毒编码超过11种不同的跨膜蛋白，包括尾锚定蛋白家族的成员。HSV1编码三种尾锚定蛋白pUL34、pUL56和pUS9，其特征是在其C末端有一个单一的疏水区域，在翻译后膜插入之前需要从核糖体上释放出来。Asna1/TRC40是一种ATP酶，它以受体依赖的方式将尾锚定蛋白靶向内质网。细胞生物学数据表明Asna1/TRC40在尾锚定蛋白生物合成中起关键和普遍作用。通过这项研究，我们旨在确定尾锚定插入机制对HSV1感染的重要性。
为了确定蛋白质-蛋白质相互作用，应用了酵母双杂交系统。使用RNA干扰使Asna1/TRC40缺失。应用瞬时转染和病毒感染实验，随后进行间接免疫荧光分析，以分析病毒蛋白的定位以及Asna1/TRC40缺失对病毒感染的影响。
所有HSV1尾锚定蛋白都与Asna1/TRC40特异性结合，但独立定位于其靶膜。虽然对细胞活力不是必需的，但Asna1/TRC40是HSV1有效复制所必需的。我们表明，复制周期的早期事件，如病毒粒子进入和整体病毒基因表达，不受Asna1/TRC40缺失的影响。此外，形成了等量的感染性病毒粒子并保持与细胞相关。这表明需要必需的尾锚定蛋白pUL34的衣壳核输出以及形成感染性病毒粒子的二次包裹都成功完成。尽管病毒生命周期的大部分过程正常进行，但病毒繁殖减少了10倍以上。我们表明，Asna1/TRC40的缺失特别影响感染后期将感染性病毒粒子释放到细胞外环境的步骤。
在疱疹病毒感染的后期，Asna1/TRC40是将成熟病毒粒子有效释放到细胞外环境所必需的。这项研究揭示了解码新形成病毒粒子胞吐作用的新工具以及迄今为止未知的抗病毒治疗细胞靶点。
