In vivo structure and dynamics of the SARS-CoV-2 RNA genome

Yan Zhang,Shu Shi,Grzegorz Kudla,Meilin Jin,Ping Li,Youzhi Mao,Jian You Lau,Youliang Wang,Wenlong Shen,Kun Huang,Hongguang Ren,Dejian Xie,Zhong Zou,Dong Wang,Zhihu Zhao

doi:10.1038/s41467-021-25999-1

Abstract

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5′-UTR and 3′-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies.

Highlights

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown
We developed a simplified SPLASH protocol to capture RNA–RNA interactions in the SARS-CoV-2 virus
Samples were collected from different phases of the SARS-CoV-2 virus life cycle to infer the dynamic structure of viral RNA at different stages

Summary

Introduction

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies. Ziv et al.[16] discovered networks of both gRNA and subgenomic RNA (sgRNA) interactions by applying specific probes to pull down each RNA species in cells, and Cao et al.[17] reconstructed structures in virions. All of these experiments were performed in a specific stage of the virus life cycle. There is need to directly compare structures from different stages to investigate their dynamics and functional relevance during the whole life cycle

Methods

Results

Conclusion