Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1
Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1
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DOI：
10.1128/jvi.00306-22
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发表时间：
2022-04
影响因子：
5.4
通讯作者：
Fumio Maeda;Akihisa Kato;Kosuke Takeshima;Misato Shibazaki;Ryota Sato;Takuma Shibata;K. Miyake;H. Kozuka-Hata;M. Oyama;Eigo Shimizu;S. Imoto;S. Miyano;S. Adachi;T. Natsume;K. Takeuchi;Yuhei Maruzuru;Naoto Koyanagi;Arii Jun;Kawaguchi Yasushi
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作者：
Fumio Maeda;Akihisa Kato;Kosuke Takeshima;Misato Shibazaki;Ryota Sato;Takuma Shibata;K. Miyake;H. Kozuka-Hata;M. Oyama;Eigo Shimizu;S. Imoto;S. Miyano;S. Adachi;T. Natsume;K. Takeuchi;Yuhei Maruzuru;Naoto Koyanagi;Arii Jun;Kawaguchi Yasushi
鉴定与病毒效应蛋白相互作用并在重要病毒过程中起作用的细胞蛋白，对于增进我们对各种病毒过程机制的理解是必要的。在本研究中，我们建立了一个新的系统，包括对单纯疱疹病毒1型（HSV - 1）核出芽复合物（NEC）进行相互作用组筛选，然后进行功能缺失筛选，以针对所鉴定的假定的与NEC相互作用的细胞蛋白，检测HSV - 1核出芽的缺陷。
摘要：本研究开发了一个由两轮筛选参与单纯疱疹病毒1型（HSV - 1）核出芽的细胞蛋白组成的系统。利用该系统，我们首先通过串联亲和纯化结合基于质谱的蛋白质组学技术，在HSV - 1感染的细胞中筛选与由HSV - 1中的UL34和UL31组成的核出芽复合物（NEC）相互作用的细胞蛋白，NEC对HSV - 1的核出芽至关重要。接下来，我们在荧光显微镜下使用针对在第一次蛋白质组学筛选中鉴定的细胞蛋白的单向导RNA，对活的HSV - 1感染的报告细胞进行基于CRISPR/Cas9的筛选，以检测核纤层相关蛋白emerin的错误定位，这是HSV - 1核出芽缺陷的一种表型。本研究聚焦于一种细胞孤儿转运蛋白SLC35E1，它是由筛选系统鉴定的细胞蛋白之一。SLC35E1的敲除降低了HSV - 1的复制，并诱导在核膜（NM）附近包含核周包膜病毒粒子（PEV）的膜内陷，在内、外核膜之间的核周间隙以及内陷结构中PEV的异常积累，以及NEC的错误定位。这些效应与先前报道的HSV - 1蛋白的突变以及对HSV - 1去包膜（HSV - 1核出芽所需的步骤之一）重要的细胞蛋白的耗竭所产生的效应相似。我们新建立的筛选系统使我们能够鉴定一种对HSV - 1有效去包膜所必需的新型细胞蛋白。
重要性：鉴定与病毒效应蛋白相互作用并在重要病毒过程中起作用的细胞蛋白，对于增进我们对各种病毒过程机制的理解是必要的。在本研究中，我们建立了一个新的系统，包括对单纯疱疹病毒1型（HSV - 1）核出芽复合物（NEC）进行相互作用组筛选，然后进行功能缺失筛选，以针对所鉴定的假定的与NEC相互作用的细胞蛋白，检测HSV - 1核出芽的缺陷。这个新建立的系统将孤儿转运蛋白SLC35E1鉴定为对HSV - 1有效去包膜所必需的一种新型细胞蛋白，为了解这一病毒过程所涉及的机制提供了见解。
The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. ABSTRACT This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.