Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
Avian Diseases Laboratory, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea.
FASEB J. 2021 Jun;35(6):e21630. doi: 10.1096/fj.202002006RR.
The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.
酸性核磷蛋白 32 家族成员 A（ANP32A）是一种细胞宿主因子，决定了禽流感病毒（AIV）的病毒聚合酶（vPol）的宿主嗜性。与人类 ANP32A（hANP32A）相比，鸡 ANP32A 含有从氨基酸残基 149-175（27 个残基）重复的额外 33 个氨基酸残基（176-208），表明这些残基可能通过加强 ANP32A 与 vPol 之间的相互作用来增加 vPol 活性。然而，hANP32A 中这 27 个残基在 AIV vPol 活性中的分子相互作用和功能作用尚不清楚。在这里，我们基于与其他人类（h）ANP32 家族成员的比较，研究了 hANP32A 的 27 个残基的功能作用。值得注意的是，与 hANP32A 和 hANP32B 不同，尽管 hANP32C 与 hANP32A 的序列同一性高于 hANP32B，但 hANP32C 不能支持 AIVs 的 vPol 活性或复制。hANP32A 与 hANP32C 之间的成对比较表明，天冬氨酸 149（D149）和天冬氨酸 152（D152）分别参与氢键和静电相互作用，从而支持 vPol 活性。这些残基的突变降低了 hANP32A 与 vPol 的相互作用。最后，我们证明了通过 CRISPR/Cas9 系统使用同源定向修复对鸡 ANP32A 内的鉴定残基进行精确替换，导致鸡细胞中的病毒复制明显减少。这些结果增加了我们对 ANP32A 功能的理解，并可能通过对 ANP32A 内的残基进行精确修饰，促进抗 AIV 鸡的开发。
