RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses

Ramya Rangan; Ivan N. Zheludev; Rhiju Das

The 5´ UTR includes five confident stem-loop structures (SL1-SL5), with structures verified by chemical mapping experiments in related coronaviruses. 9, 16 SL1 and SL2 are conserved across betacoronaviruses, with SL2 having the highest sequence conservation across the 5´ UTR. 13 The high A-U base-pairing content in the SARS-CoV-2 SL1 sequence and the bulged nucleotides align with prior reports that SL1 is relatively thermodynamically unstable to allow for the formation of longrange interactions. 23 SL2 has been shown to be critical for subgenomic RNA synthesis, with mutations in its conserved pentaloop retaining the production of genome-sized RNA, but not subgenomic RNA segments. 24 SL3, conserved only in betacoronaviruses, presents the transcriptionregulating sequence (TRS) that base pairs with one of several complementary sequences in nascent negative-sense strands in a 'copy-choice mechanism' that gives rise to discontinuous transcription of subgenomic mRNAs. 6 SL4 contains a short upstream ORF, here labeled uORF, which precedes the first longer ORF1ab of the genome. The uORF leads to attenuated transcription of ORF1ab that appears helpful but is not essential for viral replication. 13 SL5 has been implicated in a potential role in viral packaging, and contains the AUG start codon for long ORF1ab which encodes the viral replicase/transcriptase polyprotein. The SARS-CoV-2 SL5 domain has common features with the domain in other group IIb betacoronaviruses, for instance including UUCGU pentaloops on SL5a and SL5b, and a GNRA tetraloop on SL5c. 9 Prior DMS-probing data for Stem 5 in SARS-CoV aligned with the proposed SL5a,b,c structures. 9 Two additional stems (SL6 and SL7) are predicted from computer modeling here, but prior literature has not established whether such stems embedded in the coding region are functionally important across betacoronaviruses.