Meng Hailan, Wang Qi, Liu Meiling, Li Ziwei, Hao Xiaojing, Zhao Di, Dong Yaqin, Liu Shuang, Zhang Feng, Cui Jin, Ni Bo, Shan Hu, Liu Fuxiao
College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China.
Department of Animal Medicine, Shandong Vocational Animal Science and Veterinary College, Weifang, China.
Front Microbiol. 2022 Aug 17;13:957849. doi: 10.3389/fmicb.2022.957849. eCollection 2022.
Senecavirus A (SVA) is an emerging picornavirus. Its genome is one positive-sense, single-stranded RNA. The viral protein (VPg) is covalently linked to the extreme 5' end of the SVA genome. A complex hairpin-pseudoknot-hairpin (HPH) RNA structure was computationally predicted to form at the 5' end of the SVA genome. A total of three extra "U" residues (UUU) served as a linker between the HPH structure and the VPg, causing putative UUU-HPH formation at the extreme 5' end of the SVA genome. It is unclear how the UUU-HPH structure functions. One SVA cDNA clone (N0) was constructed previously in our laboratory. Here, the N0 was genetically tailored for reconstructing a set of 36 modified cDNA clones (N1 to N36) in an attempt to rescue replication-competent SVAs using reverse genetics. The results showed that a total of nine viruses were successfully recovered. Out of them, five were independently rescued from the N1 to N5, reconstructed by deleting the first five nucleotides (TTTGA) one by one from the extreme 5' end of N0. Interestingly, these five viral progenies reverted to the wild-type or/and wild-type-like genotype, suggesting that SVA with an ability to repair nucleotide defects in its extreme 5' end. The other four were independently rescued from the N26 to N29, containing different loop-modifying motifs in the first hairpin of the HPH structure. These four loop-modifying motifs were genetically stable after serial passages, implying the wild-type loop motif was not a high-fidelity element in the first hairpin during SVA replication. The other genetically modified sequences were demonstrated to be lethal elements in the HPH structure for SVA recovery, suggesting that the putative HPH formation was a crucial -acting replication element for SVA propagation.
A组赛内卡病毒（SVA）是一种新出现的小RNA病毒。其基因组为一条正链单链RNA。病毒蛋白（VPg）与SVA基因组的5′末端共价连接。通过计算预测在SVA基因组的5′末端会形成一个复杂的发夹-假结-发夹（HPH）RNA结构。共有三个额外的“U”残基（UUU）作为HPH结构与VPg之间的连接物，导致在SVA基因组的5′末端形成推测的UUU-HPH结构。目前尚不清楚UUU-HPH结构是如何发挥作用的。我们实验室之前构建了一个SVA cDNA克隆（N0）。在此，对N0进行基因改造，以构建一组36个修饰的cDNA克隆（N1至N36），试图利用反向遗传学拯救具有复制能力的SVA。结果显示，共成功拯救出9种病毒。其中，有5种是从N1至N5独立拯救出来的，它们是通过从N0的5′末端逐个删除前五个核苷酸（TTTGA）而构建的。有趣的是，这5个病毒后代恢复为野生型或/和野生型样基因型，表明SVA具有修复其5′末端核苷酸缺陷的能力。另外4种是从N26至N29独立拯救出来的，它们在HPH结构的第一个发夹中含有不同的环修饰基序。这4个环修饰基序在连续传代后基因稳定，这意味着野生型环基序在SVA复制过程中不是第一个发夹中的高保真元件。其他基因修饰序列被证明是HPH结构中SVA拯救的致死元件，这表明推测的HPH形成是SVA传播的关键作用复制元件。
