In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA–protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5′-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5′-gUUYCGUc-3′ RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5′-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5′-agc-3′/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5′-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5′-gUUUCGUc-3′ motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

中文翻译：

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结构系统发育分析揭示了所有冠状病毒中的谱系特异性 RNA 重复结构基序以及 SARS-CoV-2 的相关变异

在许多单链 (ss) RNA 病毒中，赋予选择性基因组包装的顺式包装信号通常包含短结构的 RNA 重复序列。这些结构单元，称为重复结构基序 (RSM)，可能通过特定的 RNA-蛋白质相互作用介导衣壳组装。然而，关于正义 ssRNA 冠状病毒 (CoVs) 中 RSM 的保守性和/或多样性的一般知识是有限的。通过进行结构系统发育分析，我们在几乎所有 CoV 基因组 RNA 中鉴定了多种 RSM，它们仅位于 5'-非翻译区 (UTR) 和/或多蛋白 1ab 编码序列的域间区域。一种特定于血统的方式。在所有 alpha- 和 beta-CoVs 中，除了 Embecovirus spp，显示保守六环序列的 5'-gUUYCGUc-3' RSM 的 2 到 4 个拷贝通常在位于基因组 RNA 的 5'-UTR 的茎环 5 (SL5) 中鉴定。然而，在 Embecovirus spp.中，在开放阅读框 (ORF) 1ab 中的非结构蛋白 (NSP) 3 和/或 NSP15 的编码区中发现了 2 到 8 个 5'-agc-3'/guAAu RSM 拷贝。在 gamma- 和 delta-CoVs 中，在 5'-UTR 和/或 ORF1ab 的几个簇状结构元素中发现了其他类型的 RSM。对所有 CoV 中包含 RSM 的结构元素的鉴定表明，这些 RNA 元素在 CoV 的生命周期中发挥着重要作用。在最近出现的 SARS-CoV-2 中，在其 SL5 中也发现了 β-CoV 特异性 RSM，显示了两个副本 5'-gUUUCGUc-3' 基序。然而，多序列比对显示，大多数 SARS-CoV-2 都具有带有 SL5b C241U 的变体 RSM，有趣的是，病毒蛋白的编码序列中存在多种变异，例如 Nsp12 P323L、S 蛋白 D614G 和 N 蛋白 R203K-G204R，与这种变体 RSM 同时发现。总之，对 RSM 的全面探索揭示了对整个 CoV 中 RNA 结构元素的系统发育见解，并为目前在 SARS-CoV-2 中发现的变异提供了新的视角。