SUMMARYSARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs or vaccines with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on predicted RNA structures in SARS-CoV-2 and most putative regulatory sequences are uncharacterized. Here we report the secondary structure of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq). Our results reveal previously undescribed structures within critical regulatory elements such as the genomic transcription-regulating sequences (TRSs). Contrary to previous studies, our in-cell data show that the structure of the frameshift element, which is a major drug target, is drastically different from prevailing in vitro models. The genomic structure detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

Human immunodeficiency virus-1 (HIV-1) is a retrovirus with a 10-kb single-stranded RNA genome. HIV-1 must express all of its gene products from the same primary transcript, which undergoes alternative splicing to produce diverse protein products, including structural proteins and regulatory factors 1 , 2 . Despite the critical role of alternative splicing, the mechanisms driving splice-site choice are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements 3 . We use DMS-MaPseq to probe the structure of HIV-1 RNA in cells and develop an algorithm called D etection of R NA folding E nsembles using E xpectation- M aximization (DREEM), which reveals alternative conformations assumed by the same RNA sequence. Contrary to previous models, which analyzed population averages 4 , our results reveal the widespread heterogeneous nature of HIV-1 RNA structure. In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev Responsive Element (RRE) exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis 5 – 7 that RNA conformation heterogeneity regulates splice site usage and viral gene expression.

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

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