Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (7): 2391-2406.DOI: 10.1016/j.jia.2024.04.019

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A型塞尼卡病毒内部核糖体进入位点茎环亚域的替换: 对病毒拯救的影响


  

  • 收稿日期:2023-10-18 接受日期:2024-03-09 出版日期:2024-07-20 发布日期:2024-07-02

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Qianqian Wang1, 2*, Jie Wang1*, Lei Zhang3, Xiaoxiao Duan4, Lijie Zhu1, Youming Zhang5, Yan Li4#, Fuxiao Liu1#
  

  1. 1 College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China
    2 College of Veterinary Medicine, Inner Mongolia Agricultural University, Huhhot 010018, China
    3 Shandong New Hope Liuhe Group Co., Ltd., Qingdao 266100, China
    4 Qingdao Center for Animal Disease Control & Prevention, Qingdao 266199, China
    5 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China
  • Received:2023-10-18 Accepted:2024-03-09 Online:2024-07-20 Published:2024-07-02
  • About author:Qianqian Wang, E-mail: wqq107323@163.com; # Correspondence Yan Li, E-mail: liyanqd2008@163.com; Fuxiao Liu, E-mail: laudawn@126.com * These authors contributed equally to this study
  • Supported by:
    This work was supported by the National Natural Science Foundation of China (32273000), the Qingdao Demonstration Project for People-benefit from Science and Techniques, China (23-2-8-xdny-14- nsh and 24-2-8-xdny-4-nsh), the National Program of Undergraduate Innovation and Entrepreneurship, China (202310435039), and the Open Project Fund of State Key Laboratory of Microbial Technology, China (M2023-03).

摘要:

A型塞尼卡病毒(Senecavirus A, SVA)基因组为单股正链RNA,其5' 非编码区包含一个内部核糖体进入位点(Internal ribosome entry site, IRES),由十个较大或较小的茎环结构(包括一个假结)组成,这些结构已被证明具有很好的保守性。然而,目前尚不清楚每个茎环亚域,如单个茎或环,是否也高度保守。为阐明此问题,我们通过在IRES上进行定点突变(SDM)构建了一组29个SVA cDNA克隆。SDM修饰包括:(1)茎形成的互补序列相互交换;(2)环颠换;(3)环转换;以及(4)点突变。将所有cDNA克隆分别转染细胞以拯救活病毒,但只有四个SVA拯救成功,并在二十代传代过程中具有遗传稳定性,其中一个子代的生长速度明显较慢。双荧光素酶报告试验显示,SDM修饰的IRESes均未显著抑制IRES活性。我们先前研究发现,如果完全突变十个茎结构中任一个的单一基序,均会导致病毒拯救失败。有趣的是,本研究发现SVA可独立允许三个茎结构,其各自的互补序列相互交换,同时还可以容忍一个顶端环完全转换。因此,本研究表明,并非每个茎环结构的构象都严格保守。我们之前的研究还发现,两个SVA株可通过选择复制机制在IRES区域发生重组。尽管SVA IRES的序列高度保守,但这两项研究都表明其中某些元件仍然是可变的,这为IRES与多种因子间相互作用提供了新的研究方向。

Abstract:

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.


Key words: SVA ,  HCV IRES ,  HCV-like IRES ,  Stem–loop structure ,  cDNA clone ,  virus rescue ,  mutation