Fujimoto Yoshikazu, Ozaki Kinuyo, Ono Etsuro
Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Joint Graduate School of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
J Virol. 2025 Jan 31;99(1):e0184024. doi: 10.1128/jvi.01840-24. Epub 2024 Nov 21.
Acidic nuclear phosphoprotein 32 family member A (ANP32A) is an important host factor that supports the efficient replication of avian influenza viruses (AIVs). To develop an antiviral strategy against Gs/Gd-lineage H5 highly pathogenic avian influenza (HPAI) viruses in chickens, we established chicken ANP32-knockout (chANP32A-KO) DF-1 cells and evaluated their antiviral efficacy through validation. The replication of all HPAI viruses tested in chANP32A-KO cells was significantly lower compared to that of wild-type DF-1 cells. However, when HPAI strains A/mountain hawk-eagle/Kumamoto/1/2007 (H5N1; MHE) and A/chicken/Aichi/2/2011 (H5N1; H5Aichi) were passed in chANP32A-KO cells, mutant viruses were generated, which exhibited comparable replication levels in both chANP32A-KO and wild-type DF-1 cells. Sequence analysis revealed that mammalian-adaptive amino acid mutations PB2_D256G and PA_T97I were present in the MHE mutant virus, and the PB2_E627K mutation was identified in the H5Aichi mutant virus. These mutations have also been reported to enhance the polymerase activity of AIVs in mammalian cells; however, the minigenome assay in the present study showed that the polymerase activity of mutant viruses in chANP32A-KO cells was not restored to levels comparable to those in wild-type DF-1 cells. These findings suggest that ANP32A-independent viral replication may induce amino acid substitutions associated with mammalian adaptation in AIVs. They also imply that the high efficiency of viral replication mediated by these amino acid mutations may not result from enhanced polymerase activity but rather involve other undefined mechanisms.IMPORTANCEDuring the host-switching of avian influenza viruses (AIVs) to mammalian hosts, introducing adaptive mutations into viral proteins is essential to ensure optimal functionality through virus-host protein interactions in mammalian cells. However, the mechanisms leading to adaptive mutations in viral proteins remain unclear. Among several host proteins that promote viral growth, acidic nuclear phosphoprotein 32 family member A (ANP32A) is known to be an important factor for efficient viral replication. Here, we generated mutant highly pathogenic avian influenza viruses capable of ANP32A-independent replication in a chicken-derived cell line. We demonstrated that several amino acid mutations found in the mutant viruses correspond to those associated with the mammalian adaptation of AIVs. These results suggest that ANP32A-independent viral replication is one of the mechanisms for introducing amino acid mutations that are reportedly involved in the mammalian adaptation of AIVs.
酸性核磷蛋白32家族成员A（ANP32A）是一种重要的宿主因子，可支持禽流感病毒（AIV）的高效复制。为了制定针对鸡中Gs/Gd谱系H5高致病性禽流感（HPAI）病毒的抗病毒策略，我们建立了鸡ANP32基因敲除（chANP32A-KO）DF-1细胞，并通过验证评估了它们的抗病毒效果。与野生型DF-1细胞相比，在chANP32A-KO细胞中测试的所有HPAI病毒的复制均显著降低。然而，当HPAI毒株A/凤头鹰/熊本/1/2007（H5N1；MHE）和A/鸡/爱知/2/2011（H5N1；H5爱知）在chANP32A-KO细胞中传代时，产生了突变病毒，这些突变病毒在chANP32A-KO细胞和野生型DF-1细胞中的复制水平相当。序列分析显示，MHE突变病毒中存在哺乳动物适应性氨基酸突变PB2_D256G和PA_T97I，在H5爱知突变病毒中鉴定出PB2_E627K突变。据报道，这些突变也增强了AIV在哺乳动物细胞中的聚合酶活性；然而，本研究中的微型基因组试验表明，chANP32A-KO细胞中突变病毒的聚合酶活性并未恢复到与野生型DF-1细胞相当的水平。这些发现表明，不依赖ANP32A的病毒复制可能会诱导AIV中与哺乳动物适应性相关的氨基酸替换。它们还暗示，由这些氨基酸突变介导的病毒复制高效率可能不是由聚合酶活性增强导致的，而是涉及其他未明确的机制。
重要性
在禽流感病毒（AIV）向哺乳动物宿主的宿主转换过程中，在病毒蛋白中引入适应性突变对于通过哺乳动物细胞中的病毒-宿主蛋白相互作用确保最佳功能至关重要。然而，导致病毒蛋白适应性突变的机制仍不清楚。在几种促进病毒生长的宿主蛋白中，酸性核磷蛋白32家族成员A（ANP32A）是高效病毒复制的重要因素。在这里，我们在鸡源细胞系中产生了能够独立于ANP32A进行复制的突变高致病性禽流感病毒。我们证明，在突变病毒中发现的几个氨基酸突变与AIV的哺乳动物适应性相关。这些结果表明，不依赖ANP32A的病毒复制是引入据报道参与AIV哺乳动物适应性的氨基酸突变的机制之一。
