College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.
International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.
Viruses. 2021 Oct 28;13(11):2175. doi: 10.3390/v13112175.
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100-120 aa, 120-140 aa, and 140-165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149-RGEGRFGSTG-158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.
非洲猪瘟病毒（ASFV）的脱氧尿苷 5'-三磷酸核苷水解酶（dUTPase）是病毒高效复制所必需的关键酶。先前的晶体学数据表明，dUTPase（E165R）可能是抑制 ASFV 复制的治疗靶点；然而，ASFV dUTPase 靶向结合位点的特异性尚不清楚。在本研究中，制备了 19 株鼠源单克隆抗体（mAb），其中 4 株 mAb 对 E165R 重组蛋白表现出抑制反应性。表位作图研究表明，E165R 具有三个主要的抗原区域：100-120aa、120-140aa 和 140-165aa。三种 mAb 通过与高度保守的 149-RGEGRFGSTG-158 氨基酸序列结合，抑制 E165R 的 dUTPase 活性。有趣的是，8F6 mAb 特异性识别 ASFV dUTPase 但不识别 Sus scrofa dUTPase，这可能是由于 motif V 区域的 F151、R153 和 F154 氨基酸的结构差异所致。总之，我们开发了抗 E165R 特异性 mAb，并鉴定了 ASFV dUTPase motif V 中的一个重要抗体结合抗原表位。本研究为靶向 ASFV dUTPase 抗原表位的 mAb 提供了全面的分析，可能有助于开发新型基于抗体的 ASFV 治疗药物。
