Zhang Yuan, Guo Xing, Yu Mengmeng, Sun Liuke, Qu Yuxing, Guo Kui, Hu Zhe, Liu Diqiu, Zhang Haili, Wang Xiaojun
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
Virol Sin. 2023 Oct 27;38(6):951-60. doi: 10.1016/j.virs.2023.10.009.
Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses. To date, the molecular properties of equine ANP32 (eqANP32) protein are poorly understood, particularly the mechanisms that affect equine influenza virus (EIV) RNA polymerase activity. Here, we found that there are six alternative splicing variants of equine ANP32A (eqANP32A) with different levels of expression. Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees, with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity. Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation, but did not affect their ability to bind RNA polymerase. We also demonstrated that there is only one transcript of eqANP32B, and that this transcript showed only very low support to the EIV RNA polymerase. This functional defect in eqANP32B is caused by the sequence of the 110-259 amino acids at its C-terminus. Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses. In conclusion, our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B, suggesting for the first time that the N-cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.
宿主ANP32家族蛋白对于维持流感病毒RNA聚合酶的活性至关重要，并且在流感病毒的跨物种传播中发挥重要作用。迄今为止，马ANP32（eqANP32）蛋白的分子特性了解甚少，尤其是影响马流感病毒（EIV）RNA聚合酶活性的机制。在此，我们发现马ANP32A（eqANP32A）有六种可变剪接变体，其表达水平各不相同。进一步研究表明，eqANP32A的这六种剪接变体对EIV RNA聚合酶的活性有不同程度的支持，其中变体eqANP32A_X2表达丰度最高，对聚合酶活性的支持也最高。序列分析表明，六种剪接变体的N端帽区差异显著影响其N端构象，但不影响它们与RNA聚合酶结合的能力。我们还证明eqANP32B只有一种转录本，并且该转录本对EIV RNA聚合酶的支持非常低。eqANP32B的这种功能缺陷是由其C端110 - 259个氨基酸的序列引起的。我们的结果表明，主要是eqANP32A_X2蛋白决定了EIV在马体内的复制效率。总之，我们的研究剖析了eqANP32家族蛋白的分子特性，揭示了eqANP32A和eqANP32B的序列特征，首次表明ANP32A蛋白的N端帽区在支持流感病毒聚合酶活性方面也发挥重要作用。
