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Abstract：
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.
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.
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.
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.
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.
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.
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.
摘要：
背景：戊型肝炎病毒（HEV）的一个特殊特征是其依赖外泌体途径释放病毒。基因组复制是通过病毒多蛋白PORF1介导的，但对其亚细胞定位知之甚少。
方法：pORF1及其亚结构域的亚细胞定位，根据病毒复制酶的结构预测产生和克隆，通过共聚焦激光扫描显微镜进行分析。通过超速离心分离从细胞释放的外来体，并通过等密度密度梯度离心进行分析。随后进行荧光测定或蛋白质印迹分析或RT-qPCR以更详细地分析分离的颗粒。
结果：我们发现pORF1在内体系统内积累，最主要的是MVB。多蛋白的七个亚结构域的表达表明，PCP（木瓜蛋白酶样半胱氨酸蛋白酶）是唯一像全长蛋白一样定位的结构域。PCP缺陷型PORF1突变体失去了与MVB的关联。引人注目的是，pORF1和PCP均可通过外泌体释放。同样，在缺乏PORF2/3的情况下，基因组RNA仍然通过外泌体释放。
结论：总之，我们发现pORF1以依赖PCP的方式定位到MVB，然后是外泌体释放。这揭示了HEV生命周期的新方面，因为复制和释放可以在内体界面耦合。此外,这可能介导衣壳非依赖性传播或可能促进病毒感染的传播，因为在从头感染期间进入细胞的基因组很容易遇到外来转移的PORF1。
方法：pORF1及其亚结构域的亚细胞定位，根据病毒复制酶的结构预测产生和克隆，通过共聚焦激光扫描显微镜进行分析。通过超速离心分离从细胞释放的外来体，并通过等密度密度梯度离心进行分析。随后进行荧光测定或蛋白质印迹分析或RT-qPCR以更详细地分析分离的颗粒。
结果：我们发现pORF1在内体系统内积累，最主要的是MVB。多蛋白的七个亚结构域的表达表明，PCP（木瓜蛋白酶样半胱氨酸蛋白酶）是唯一像全长蛋白一样定位的结构域。PCP缺陷型PORF1突变体失去了与MVB的关联。引人注目的是，pORF1和PCP均可通过外泌体释放。同样，在缺乏PORF2/3的情况下，基因组RNA仍然通过外泌体释放。
结论：总之，我们发现pORF1以依赖PCP的方式定位到MVB，然后是外泌体释放。这揭示了HEV生命周期的新方面，因为复制和释放可以在内体界面耦合。此外,这可能介导衣壳非依赖性传播或可能促进病毒感染的传播，因为在从头感染期间进入细胞的基因组很容易遇到外来转移的PORF1。
