The short- and long-range RNA Interactome of SARS-CoV-2

The Coronaviridae are a family of positive- strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single stranded RNA genome in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo long-range RNA interactome of the full-length SARS-CoV-2 genome and the subgenomic mRNAs. We uncover a network of RNA-RNA interactions spanning tens of thousands of nucleotides that facilitate the unique transcription mode of coronaviruses, and reveal that the viral genome adopts alternative topologies inside cells and undergoes genome cyclization. Moreover, we discover long RNA-bridges between adjacent open reading frames that encircle the programmed ribosomal frame-shifting element, and demonstrate their conservation in vivo for the related MERS-CoV. Finally, the SARS-CoV-2 genome and subgenomic mRNAs engage in different sets of interactions with cellular RNAs. Our findings illuminate RNA-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies. We recently developed Crosslinking Of Matched RNAs And Deep Sequencing (COMRADES) for in-depth RNA conformation capture in living cells (Ziv et al., 2018). COMRADES can detect base-paired regions in RNA inside the cell, using a clickable psoralen derivative to specifically crosslink double-stranded RNA, and high throughput sequencing to retrieve the base-pairing information (Figure S1A). Following in vivo crosslinking, the viral RNA is selectively captured, fragmented and subjected to a click-chemistry reaction to add a biotin tag to crosslinked regions. Crosslinked RNA duplexes are then selectively captured using streptavidin affinity purification. Half of the resulting RNA is proximity ligated, following reversal of the crosslink to create chimeric RNA templates for high throughput sequencing. The other half is used as a control, in which reversal of the crosslink precedes the proximity ligation, and accurately represents the background level of non-specific ligation. The coupling of two enrichment steps, first of viral RNA, and second of crosslinked RNA duplexes provides high structural depth for identification of both long- and short-lived conformations. COMRADES can therefore measure (i) the structural diversity of alternative RNA conformations that coexist inside cells; (ii) short-distance, as well as long-distance (over tens of thousands of nucleotides) base-pairing within the same RNA molecule; and (iii) base-pairing between different RNA molecules, such as those of host and viral origin (Kudla et al., 2020; Ziv et al., 2018). Here we applied COMRADES to study the structural diversity of SARS-CoV-2 gRNA and sgmRNAs inside cells. Whereby we have conducted in duplicate - MERS and SARS-CoV-2 gRNA as well as MERS and SARS-CoV-2 sgmRNAs.

冠状病毒科（Coronaviridae）是一类正链RNA病毒（positive-strand RNA viruses），包含引发新型冠状病毒肺炎（COVID-19）大流行的病原体——严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）。该科病毒拥有自然界中规模最大的单链RNA基因组，其转录与复制通路高度依赖长距离RNA-RNA相互作用（long-distance RNA-RNA interactions）。本研究通过实验手段，绘制了全长SARS-CoV-2基因组及亚基因组mRNA（subgenomic mRNAs）的体内长距离RNA互作组（RNA interactome）。

我们揭示了跨越数万个核苷酸的RNA-RNA相互作用网络，该网络介导冠状病毒独特的转录模式；同时发现病毒基因组在细胞内存在多种可变拓扑构象，并会发生基因组环化。此外，我们还在相邻开放阅读框（open reading frame, ORF）之间发现了环绕程序性核糖体移码元件（programmed ribosomal frame-shifting element）的长RNA桥接结构，并证实该结构在相关的中东呼吸综合征冠状病毒（MERS-CoV）体内具有保守性。最后，我们发现SARS-CoV-2基因组与亚基因组mRNA可与宿主RNA形成不同的相互作用谱。

本研究结果阐明了调控冠状病毒复制、不连续转录与翻译的RNA依赖机制，将为未来开发抗病毒策略提供重要支撑。

我们此前开发了匹配RNA交联与深度测序（Crosslinking Of Matched RNAs And Deep Sequencing, COMRADES）技术，用于活细胞内的高分辨率RNA构象捕获（Ziv等，2018）。该技术可通过可点击补骨脂素衍生物特异性交联细胞内的双链RNA（double-stranded RNA），并结合高通量测序（high throughput sequencing）获取碱基配对信息（图S1A）。具体流程如下：完成体内交联后，我们选择性捕获病毒RNA并进行片段化，随后通过点击化学反应（click-chemistry reaction）为交联区域添加生物素标签（biotin tag）；再利用链霉亲和素亲和纯化（streptavidin affinity purification），选择性富集交联的RNA双链体（RNA duplexes）。将所得RNA分为两组：一组先进行近端连接（proximity ligation），待交联逆转后生成嵌合RNA模板（chimeric RNA templates），用于高通量测序；另一组作为对照，先逆转交联再进行近端连接，可准确反映非特异性连接（non-specific ligation）的背景水平。两次富集步骤（先富集病毒RNA，再富集交联RNA双链体）可实现高结构分辨率，从而能够识别瞬时存在与稳定存在的RNA构象。

因此，COMRADES技术可用于：(i) 检测细胞内共存的多种RNA构象的结构多样性；(ii) 检测同一RNA分子内的短距离及长距离（跨越数万个核苷酸）碱基配对；(iii) 检测不同RNA分子间的碱基配对，例如宿主与病毒来源的RNA分子（Kudla等，2020；Ziv等，2018）。

本研究将COMRADES技术应用于活细胞内SARS-CoV-2基因组RNA（genomic RNA, gRNA）及亚基因组mRNA（sgmRNA）的结构多样性研究。实验设置两组生物学重复，分别针对MERS-CoV与SARS-CoV-2的基因组RNA（gRNA），以及二者的亚基因组mRNA（sgmRNA）开展实验。