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

Abstract

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 genomes 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 RNA-RNA interactome of the full-length SARS-CoV-2 genome and its subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome adopts alternative topologies inside cells and undergoes genome cyclization. In addition, the SARS-CoV-2 genome and subgenomic mRNAs engage in different interactions with host RNAs. Most importantly, we discovered a long-range RNA-RNA interaction - the FSE-arch - that encircles the programmed ribosomal frame-shifting element. The FSE-arch is conserved in the related MERS-CoV virus and is under purifying selection. Our findings illuminate RNA-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.

Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function

“…Prior scans for positive selection within the SARS-CoV-2 genome used elevated ω as the signal of positive selection, which restricts attention to positive selection based on changes in protein function. For coronaviruses this is a notable limitation, given that many aspects of the lifecycle involve RNA function ( Madhugiri et al, 2016 ; Ziv et al, 2020 ; Alhatlani, 2020 ). In addition, the secondary structure of some segments within the RNA genome is well conserved among coronavirus species, which implies a functional role ( Rangan et al, 2020 ; Sanders et al, 2020 ; Huston et al, 2020 ).…”

“…These functions could, in principle, affect genome or transcript function, or both. Plausible possibilities include secondary structures that recruit specific RNA-binding proteins to mediate transcriptional regulation or transcript processing ( Pirakitikulr et al, 2016 ; Pan et al, 2020 ), that mediate looping for other reasons ( Gebhard, Filomatori & Gamarnik, 2011 ; Ziv et al, 2020 ), or that simply facilitate or impede processivity of the replication or translation machinery ( MacFadden et al, 2018 ).…”

Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function

“…Prior scans for positive selection within the SARS-CoV-2 genome used elevated ω as the signal of positive selection, which restricts attention to positive selection based on changes in protein function. For coronaviruses this is a notable limitation, given that many aspects of the lifecycle involve RNA function (Madhugiri et al, 2016; Ziv et al, 2020; Alhatlani, 2020). In addition, the secondary structure of some segments within the RNA genome is well conserved among coronavirus species, which implies a functional role (Rangan et al, 2020; Sanders et al, 2020; Huston et al, 2020a).…”

The stem loop 2 motif is a site of vulnerability for SARS-CoV-2

“…1B) in a 2.7 Å resolution crystal structure (Robertson et al, 2005). Experimental mapping of RNA-RNA interactions of the positive sense SARS-CoV-2 viral species, probed within the host cell, is consistent with the s2m element being structured, and possibly pairing with open reading frame ORF1a at some stage (Ziv et al, 2020).…”