A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V¹⁰⁵DLP¹⁰⁸ Immunoactivity

The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region.戊型肝炎病毒（hepatitis E virus, HEV）是全球范围内引发重症病毒性肝炎的致病原。动物被视为HEV的储存宿主，尤以猪类最为典型。尽管猪与犬的HEV感染通常无明显症状，但在发展中国家，该病毒会导致合并慢性基础肝病的患者以及孕妇出现极高的死亡率。戊型肝炎病毒开放阅读框2（open reading frame 2, ORF2）常被用作诊断靶点，用于检测血清样本中针对HEV的特异性抗体。近年来的研究进一步证实，ORF3蛋白亦可作为血清抗HEV检测的潜在靶点。然而，ORF3蛋白的表位分布特征仍尚不明确。本研究证实，4型HEV ORF3的羧基末端存在连续氨基酸基序VDLP，该序列是ORF3蛋白表位的核心区域。此外，其免疫活性依赖于与上游元件的协同相互作用。上游富脯氨酸结构域中的三个脯氨酸残基（P99、P102与P103）对VDLP的免疫活性具有显著调控作用。酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）结果显示，包含已鉴定VDLP表位的肽段SAPPLPPVVDLP与SAPPLPPVVDLPQLGL，与抗HEV血清的反应强度弱于商用ELISA试剂盒。本研究的综合发现为HEV ORF3的表位分布特征提供了极具价值的参考信息，并加深了我们对富脯氨酸结构域如何影响C端区域下游氨基酸免疫活性的理解。