Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

Abstract

<jats:p>SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.</jats:p>

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

“…Due to its importance in the life cycle of many important viruses and coronaviruses in particular, programmed frameshifting has been extensively studied using a range of structural and functional approaches ( 3 ). The structure of a 3’ stimulatory pseudoknot in isolation or in context of the viral genome has been proposed recently by various groups using techniques that include molecular dynamics, nuclease mapping, in vivo SHAPE, NMR and cryo-EM ( 6 , 13 – 16 ). Furthermore, a ribosomal complex with a frameshift stimulatory pseudoknot from the avian infectious bronchitis virus was reported at low resolution ( 17 ).…”

“…The intermediate local resolution (5-7 Å) of the cryo-EM map in the area of the pseudoknot allowed us to visualize the overall fold of the RNA and readjust its previously predicted secondary structure ( 13 – 16 , 21 ) (Fig. 1E).…”

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

“…However, our data did not support the folding of the stem-loop pseudoknot at the 3′ UTR. Of note, two recent studies using SHAPE methods to map the structure of SARS-CoV-2 inside cells similarly failed to identify this pseudoknot (Huston et al, 2020;Sun et al, 2020) , raising questions regarding its stability in vivo .…”

“…For example, interactions between promoters and enhancers dictate the rate of transcription along the eukaryotic genome (Rowley and Corces, 2018) . Great effort is being made to reveal the structural landscapes of the SARS-CoV-2 genome (Andrews et al, 2020;Huston et al, 2020;Kelly et al, 2020;Lan et al, 2020;Manfredonia et al, 2020;Ryder, 2020;Sanders et al, 2020;Sun et al, 2020) .…”

Structure-Altering Mutations of the SARS-CoV-2 Frame Shifting RNA Element

“…Although it is not conclusively known what the FSE structure is in SARS-CoV-2 and whether this region's folding may be affected by neighboring sequences and its binding to the nucleocapsid protein (see Conclusion and Discussion), other recent works have used various computational tools and experimental input to suggest its structure (14,52).…”