New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle
New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle
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DOI：
10.1038/s41598-019-42737-2
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发表时间：
2019-04-18
影响因子：
4.6
通讯作者：
Cocquerel, Laurence
中科院分区：
文献类型：
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作者：
Ankavay, Maliki;Montpellier, Claire;Cocquerel, Laurence
戊型肝炎病毒(HEV)基因组编码包括ORF2衣壳蛋白在内的三种蛋白。最近，我们证明了HEV产生三种不同形式的ORF2：(I)ORF2i形式(感染性ORF2)，它是感染性颗粒的组成部分；(Ii)分泌型ORF2g(糖基化ORF2)和ORF2c(裂解ORF2)形式，它们与感染性颗粒无关，但是HEV感染患者血清中的主要抗原。ORF2蛋白序列包含三个高度保守的潜在N-糖基化位点(N1、N2和N3)。ORF2N-糖基化在HEV生命周期中的地位和生物学意义仍有待阐明。在这里，我们产生并广泛鉴定了一系列ORF2突变体，其中三个N-糖基化位点单独或联合突变。我们证明了ORF2g/c蛋白在N1和N3位点上是N-糖基化的，但在N2位点上没有糖基化。我们发现ORF2蛋白的N-糖基化在感染性HEV颗粒的复制和组装中不起任何作用。我们发现糖基化的ORF2g/c形式是非常稳定的蛋白质，是患者抗体的靶标。我们还证明了ORF2i蛋白被转位到感染细胞的细胞核中。因此，我们的研究对ORF2表达的分子机制有了新的认识。
Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.