Residues Critical for Duck Hepatitis B Virus Neutralization Are Involved in Host Cell Interaction

To date, no detailed analysis of the neutralization properties of duck hepatitis B virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate here that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizing epitopes, both inhibit virus binding to primary duck hepatocytes and neutralize virus infectivity. An extensive mutagenesis of the motif (88)WTP(90), which is the shortest sequence of the epitope recognized by the virus-neutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations within this epitope affected neither virus replication nor infectivity but abolished virus neutralization by MAb 900 completely. Interestingly, mutants with two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with significantly reduced binding to primary duck hepatocytes and could be rescued by trans complementation with wild-type pre-S protein. Taken together, these results indicate that each amino acid of the DHBV pre-S sequence (88)WTP(90) is critical for recognition by the neutralizing MAb 900 and that replacement of the first two or all three residues strongly reduces virus interaction with hepatocytes and abrogates infectivity. These data imply that the motif (88)WTP(90) contains key residues which are critical for interaction with both the neutralizing MAb and the host cell.

迄今为止，尚未见针对鸭乙型肝炎病毒（Duck Hepatitis B Virus, DHBV）中和特性的详细分析报道，目前仍不清楚已知的中和表位（neutralization epitopes）是否对应病毒受体结合位点，亦或是与病毒细胞进入通路相关的序列。本研究证实，针对两段覆盖多数DHBV前S（pre-S）中和表位序列的重叠肽段（氨基酸83至97位与93至107位），均可抑制病毒与原代鸭肝细胞（primary duck hepatocytes）结合，并中和病毒感染性。为明确参与上述相互作用的氨基酸，研究人员针对病毒中和性单克隆抗体（monoclonal antibody, MAb）900所识别的最短表位基序(88)WTP(90)开展了大规模诱变（mutagenesis）分析。该表位内的单点突变（single point mutations）既不影响病毒复制，亦不改变病毒感染性，却可完全消除单克隆抗体900对病毒的中和作用。有趣的是，该表位内连续替换两个或三个残基的突变体（SIP与SIH）虽保留了复制能力（replication competence），却不再具备感染性。SIH与SIP突变病毒颗粒的感染性丧失，与其与原代鸭肝细胞的结合能力显著降低相关，且可通过野生型前S蛋白进行反式互补（trans complementation）得以挽救。综上，本研究结果表明，DHBV前S序列(88)WTP(90)中的每一个氨基酸均对中和性单克隆抗体900的识别至关重要；替换该基序的前两个残基或全部三个残基，可大幅降低病毒与肝细胞的相互作用并消除感染性。本研究数据提示，(88)WTP(90)基序包含了与中和性单克隆抗体及宿主细胞相互作用的关键残基。