Tertiary folds of the SL5 RNA from the 5 proximal region of SARS-CoV-2 and related coronaviruses

Coronavirus genomes sequester their start codons within stem - loop 5 (SL5), a structured, 5 ' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four - way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three- dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe- acute- respiratory- syndrome- related coronavirus 2 (SARS- CoV-2), resolved at 4.7 angstrom resolution, exhibits a T- shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 & Aring; resolution, respectively) indicate that the junction geometry and inter- hexaloop distances are conserved features across these human- infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non - human- infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 & Aring; resolution). SL5s from human- infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 & Aring; resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU- capped arms, but with a phylogenetically distinct crossing angle, an X- shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross - genus similarities and notable differences, with implications for potential protein- binding modes and therapeutic targets.