Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.
Highlights
Since the outbreak of SARS-CoV-2 in December 2019, the virus has infected at least 41 million individuals and caused more than 1.1 million deaths worldwide
The target SARS-CoV-2 untranslated region (UTR) minigene reporter sequence was inserted in a T7 transcription cassette
For chemically probing the structure of this in vivo transcribed viral UTR minigene transcript, the transfected BSR-T5/7 cells were transiently treated with dimethyl sulfate (DMS) at 48 h after the transfection
Summary
Since the outbreak of SARS-CoV-2 in December 2019, the virus has infected at least 41 million individuals and caused more than 1.1 million deaths worldwide. In a model β-CoV that is permissive to mice, mouse hepatitis virus (MHV), the cis-acting RNA elements at the 30 UTR were reported to be essential for viral transcription and replication [7,8]. We report the RNA structure of SARS-CoV-2 30 UTR by using a chemical probing strategy, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq) [18] in virions and minigene-transfected cells. DMS-MaPseq has previously been used to elucidate the SARS-CoV-2 RNA structures in the virus-infected cells, the 30 UTR was, not discussed in that study [11]. The chemical mechanism of DMS reaction with RNA is different from another commonly used chemical probing strategy, namely selective 20 -hydroxyl acylation analyzed by primer extension (SHAPE), which uses activated esters or amides to acylate the 20 -OH group on the ribose regardless of the nucleobase identity [19]. Cs but not Gs and Us (see Figure A1 for the DMS-MaPseq workflow)
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