Data_Sheet_1_Processing and Subcellular Localization of the Hepatitis E Virus Replicase: Identification of Candidate Viral Factories.doc

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non–structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

甲型肝炎病毒（HEV）是全球急性肝炎的主要病因。HEV是一种正链RNA病毒，表达三个开放阅读框（ORFs）。ORF1编码ORF1非结构多聚蛋白，该蛋白是病毒的复制酶，可转录全长基因组以及编码结构蛋白ORF2和ORF3的亚基因组RNA。本研究聚焦于复制步骤，旨在确定ORF1多聚蛋白在HEV生命周期中是否被加工，以及复制在宿主细胞内的具体位置。鉴于无商业抗体可识别HEV复制细胞中的ORF1，本研究旨在在不影响病毒复制效力的前提下，在ORF1蛋白中插入表位标签。因此，在高度可变区域选择了两个插入位点，以容纳V5表位，同时保留HEV的复制效力。一旦整合到感染性的全长Kernow C-1 p6菌株中，V5表位对基因组RNA的复制以及亚基因组RNA的产生均无影响。此外，V5标记的病毒颗粒与野生型颗粒一样，对Huh-7.5细胞具有感染性。接下来，在异源表达和复制性HEV系统中，比较了V5标记的ORF1的表达模式。在电穿孔后的第25天，检测到所有三个系统中均表达的分子量较高的蛋白（180 kDa），该蛋白很可能对应于未经加工的ORF1形式。此外，在细胞质和细胞核内均检测到分子量较低且较少量的产物。同时，通过共聚焦显微镜，V5标记的ORF1被定位在细胞核内，也作为紧密的核周亚结构存在，其中检测到ORF2和ORF3蛋白。重要的是，利用原位杂交（RNAScope®）技术，在HEV产生细胞的核周亚结构中定位到正链和负链的HEV RNA。最后，通过同时检测这些亚结构中的HEV基因组RNA和病毒蛋白，我们确定了候选的HEV工厂。