Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Although the essentiality of HEV nonstructural polyprotein (pORF1) putative Y-domain (Yd) has been established in viral pathogenesis, its structural-functional role remains elusive. The current research discusses the novel exploration on Yd protein expression, purification, biophysical characterization and structure-based docking analysis. The codon optimized synthetic gene and optimized expression parameters i.e., 5 h induction with 0.25 mM IPTG at 37 °C, resulted in efficient production of Yd protein (~40 kDa) in E. coli BL21(DE3) cells. Majority of the recombinant Yd (rYd) protein expressed as inclusion bodies was solubilized in 0.5% N-lauroylsarcosine and purified using Ni-NTA chromatography. Circular dichroism (CD) and UV visible absorption spectroscopic studies on Yd revealed both secondary and tertiary structure stability in alkaline range (pH 8.0–10.0), suggesting correlation with its physiological activity. Thus, loss in structure at low pH perhaps play crucial role in cytoplasmic-membrane interaction. The biophysical data were in good agreement with insilico structural analyses, which suggested mixed α/β fold, non-random and basic nature of Yd protein. Furthermore, due to Yd protein essentiality in HEV replication and pathogenesis, it was considered as a template for docking and drug-likeness analyses. The 3D modeling of Yd protein and structure-based screening and drug-likeness of inhibitory compounds, including established antiviral drugs led to the identification of top nine promising candidates. Nonetheless, in vitro studies on the predicted interaction of Yd with intracellular-membrane towards establishing replication-complexes as well as validations of the proposed therapeutic agents are warranted.

戊型肝炎病毒 (HEV) 是全球戊型肝炎感染的主要病原体。尽管 HEV 非结构多蛋白 (pORF1) 推定的 Y 域 (Yd) 的重要性已在病毒发病机制中确立，但其结构功能作用仍然难以捉摸。目前的研究讨论了对Yd蛋白表达、纯化、生物物理表征和基于结构的对接分析的新探索。密码子优化的合成基因和优化的表达参数，即在 37 °C 下用 0.25 mM IPTG 诱导 5 小时，导致在大肠杆菌中有效生产 Yd 蛋白 (~40 kDa)BL21（DE3）细胞。大多数表达为包涵体的重组 Yd (rYd) 蛋白溶解在 0.5% N-月桂酰肌氨酸中，并使用 Ni-NTA 色谱法纯化。对 Yd 的圆二色性 (CD) 和紫外可见吸收光谱研究揭示了在碱性范围 (pH 8.0-10.0) 中二级和三级结构的稳定性，表明其与其生理活性相关。因此，低 pH 值下的结构损失可能在细胞质-膜相互作用中起关键作用。生物物理数据与in silico非常吻合结构分析表明，Yd 蛋白具有混合的 α/β 折叠、非随机和碱性性质。此外，由于 Yd 蛋白在 HEV 复制和发病机制中的重要性，它被认为是对接和药物相似性分析的模板。Yd 蛋白的 3D 建模和基于结构的筛选和抑制性化合物的药物相似性，包括已建立的抗病毒药物，导致了前九个有希望的候选药物的鉴定。尽管如此，关于 Yd 与细胞内膜的预测相互作用以建立复制复合物的体外研究以及对所提出的治疗剂的验证是有必要的。