The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

IF 7.1 1区 医学 Q1 GASTROENTEROLOGY & HEPATOLOGY Cellular and Molecular Gastroenterology and Hepatology Pub Date : 2024-01-01 DOI:10.1016/j.jcmgh.2024.01.001
Mirco Glitscher, Inga Mareike Spannaus, Fabiane Behr, Robin Oliver Murra, Kathrin Woytinek, Daniela Bender, Eberhard Hildt
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Abstract

Background

A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization.

Methods

Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase–polymerase chain reaction to analyze separated particles in more detail.

Results

We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein’s 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3.

Conclusions

Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.

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HEV pORF1中的蛋白酶结构域介导了复制酶在多囊体中的定位及其外泌体释放
背景戊型肝炎病毒(HEV)的一个特点是依赖外泌体途径释放病毒。方法通过激光共聚焦扫描显微镜分析了根据病毒复制酶的结构预测生成并克隆的 pORF1 及其亚域的亚细胞定位。通过超速离心法分离出细胞中释放的外泌体,并用等容密度梯度离心法进行分析。结果我们发现 pORF1 在内含体系统中积聚,主要积聚在 MVB 中。表达多聚蛋白的七个亚结构域发现,PCP(木瓜蛋白酶样半胱氨酸蛋白酶)是唯一与全长蛋白一样定位的结构域。缺乏 PCP 的 pORF1 突变体失去了与 MVB 的联系。令人吃惊的是,pORF1 和 PCP 都能通过外泌体释放。结论综上所述,我们发现 pORF1 以 PCP 依赖性方式定位到 MVB,然后通过外泌体释放出来。这揭示了 HEV 生命周期的新方面,因为复制和释放可能在内体界面上耦合。此外,这可能会介导不依赖于囊膜的传播,也可能会促进病毒感染的传播,因为在新感染过程中进入细胞的基因组很容易遇到外泌体转移的 pORF1。
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来源期刊
CiteScore
13.00
自引率
2.80%
发文量
246
审稿时长
42 days
期刊介绍: "Cell and Molecular Gastroenterology and Hepatology (CMGH)" is a journal dedicated to advancing the understanding of digestive biology through impactful research that spans the spectrum of normal gastrointestinal, hepatic, and pancreatic functions, as well as their pathologies. The journal's mission is to publish high-quality, hypothesis-driven studies that offer mechanistic novelty and are methodologically robust, covering a wide range of themes in gastroenterology, hepatology, and pancreatology. CMGH reports on the latest scientific advances in cell biology, immunology, physiology, microbiology, genetics, and neurobiology related to gastrointestinal, hepatobiliary, and pancreatic health and disease. The research published in CMGH is designed to address significant questions in the field, utilizing a variety of experimental approaches, including in vitro models, patient-derived tissues or cells, and animal models. This multifaceted approach enables the journal to contribute to both fundamental discoveries and their translation into clinical applications, ultimately aiming to improve patient care and treatment outcomes in digestive health.
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