Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Center for Emerging, Zoonotic and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
mBio. 2024 Nov 13;15(11):e0263524. doi: 10.1128/mbio.02635-24. Epub 2024 Oct 8.
Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication , providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both and . We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.
戊型肝炎病毒（HEV）与其他嗜肝病毒不同，因为它是动物源性的。HEV-1 和 HEV-2 专门感染人类，而 HEV-3 和 HEV-4 则是动物源性的。然而，导致跨物种 HEV 传播的病毒和/或宿主因素仍不清楚。HEV 中的高变区（HVR）非常异源，与 HEV 的适应性有关。在这里，我们研究了 HVR 中丝氨酸磷酸化在 HEV 复制中的潜在作用。我们首先分析了不同 HEV 基因型的 HVR 序列，并在 HVR 的 N 端鉴定出一个独特的区域，该区域在人类特有的 HEV 基因型中是可变的，但在动物源性 HEV 基因型中相对保守。使用预测工具，我们鉴定了四个潜在的磷酸化位点，这些位点在动物源性 HEV-3 和 HEV-4 基因组中高度保守，但在人类特有的 HEV-1 株中不存在。为了探索这些假定磷酸化位点的功能意义，我们在 HEV-3 感染性克隆和指示复制子中引入突变，分别用丙氨酸或天冬氨酸替代每个丝氨酸残基，并评估这些取代对 HEV-3 复制的影响。我们发现磷酸化明显的 S711A 突变体显著降低了病毒复制，而磷酸化模拟的 S711D 突变体则适度降低了病毒复制。相反，其他三个丝氨酸残基的突变对 HEV-3 复制没有显著影响。此外，我们证明了 Ser711 磷酸化不会改变动物源性 HEV-3 的宿主细胞嗜性。总之，我们的结果表明，Ser711 残基的潜在磷酸化显著影响 HEV-3 的复制，为动物源性 HEV 传播的潜在机制提供了新的见解。
重要性：HEV 是一种重要的动物源性病原体，可引起急性和慢性戊型肝炎以及肝外疾病，如神经系统后遗症。动物源性 HEV-3 与慢性感染和神经系统疾病有关。促进跨物种 HEV 感染的特定病毒和/或宿主因素尚不清楚。ORF1 中的固有无序 HVR 对病毒适应性至关重要，
我们假设，未知宿主细胞激酶对 HVR 中丝氨酸残基的磷酸化与跨物种 HEV 传播有关。在这项研究中，我们在动物源性 HEV 株中鉴定出 HVR 中的一个保守区域，但在人类特有的 HEV-1 和 HEV-2 中不存在。我们阐明了 Ser711 残基磷酸化在动物源性 HEV-3 复制中的重要作用，而不改变宿主细胞嗜性。这些发现有助于我们理解跨物种 HEV 传播的机制。
