A conserved RNA pseudoknot in a putative molecular switch domain of the 3′-untranslated region of coronaviruses is only marginally stable

Abstract

The 39-untranslated region (UTR) of the group 2 coronavirus mouse hepatitis virus (MHV) genome contains a predicted bulged stem-loop (designated P0ab), a conserved cis-acting pseudoknot (PK), and a more distal stem-loop (designated P2). Base-pairing to create the pseudoknot-forming stem (P1 pk ) is mutually exclusive with formation of stem P0a at the base of the bulged stemloop; as a result, the two structures cannot be present simultaneously. Herein, we use thermodynamic methods to evaluate the ability of individual subdomains of the 39 UTR to adopt a pseudoknotted conformation. We find that an RNA capable of forming only the predicted PK (58 nt; 39 nucleotides 241-185) adopts the P2 stem-loop with little evidence for P1 pk pairing in 0. , 100 mM K + ), and unfolded at 37°C. Similar findings characterize an RNA 59 extended through the P0b helix only (89 nt; 294-185). In contrast, an RNA capable of forming either the P0a helix or the pseudoknot (97 nt; 301-185) forms no P1 pk helix. Thermal unfolding simulations are fully consistent with these experimental findings. These data reveal that the PK forms weakly and only when the competing doublehairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.

Coronavirus cis-Acting RNA Elements

“…They also supported base-pairing interactions upstream of the HCoV-NL63 SL2, thus supporting the formation of the predicted small hairpin in this region, while we failed to obtain experimental support for this hairpin in HCoV-229E. Also, the structure probing data did not support the formation of a stable PK structure, possibly reflecting a low thermodynamic stability as previously reported for the equivalent PK in betacoronaviruses (Stammler et al, 2011). Further experiments including reverse genetics studies are required to confirm the existence and biological significance of the predicted alphacoronavirus PK structure.…”

“…Also in this case, our knowledge of coronavirus 3 0 cis-acting elements is largely based on studies using betacoronaviruses, such as MHV. A combination of bioinformatics, biochemical analyses, and functional studies was used to identify and characterize cis-acting RNA elements in the 3 0 UTR (Goebel et al, 2004aHsue and Masters, 1997;Hsue et al, 2000;Liu et al, 2001Liu et al, , 2013Stammler et al, 2011;Williams et al, 1999;Z€ ust et al, 2008). More recently, these studies were extended to alphacoronaviruses using genus-wide bioinformatics analyses.…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…Further evidence to support this model was obtained in a subsequent MHV reverse genetics study by Liu et al (Liu et al, 2013). Thermodynamic investigations revealed a limited stability of the PK structure (Stammler et al, 2011), further supporting the structural flexibility of this cis-acting element and, thus, its proposed role as a 'molecular switch'. The region downstream of the PK is less well conserved among betacoronaviruses.…”

“…By contrast, no such interactions were seen in HCoV-229E. Also, the structure probing data did not support the formation of a stable PK structure, possibly reflecting a similarly low thermodynamic stability as determined for the equivalent PK in betacoronaviruses (Stammler et al, 2011). Further studies including reverse genetics experiments are required to confirm the existence and biological significance of the predicted alphacoronavirus PK structure.…”

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

“…Other positive-strand RNA viruses have well-described structural elements that might be able to stall and/or inhibit XRN1. These include the large stem-loop structures present in the ambisense RNA segments of arenaviruses (Pinschewer et al 2005) and pseudoknot/double hairpin structures of coronaviruses (Stammler et al 2011). Several positive-sense plant viruses also generate some apparent subgenomic stable decay products that may arise by a mechanism similar to the flavivirus sfRNA (Iwakawa et al 2008;Peltier et al 2012).…”