An Optimal cis -Replication Stem-Loop IV in the 5′ Untranslated Region of the Mouse Coronavirus Genome Extends 16 Nucleotides into Open Reading Frame 1

Abstract

cis-acting structures in positive-strand [(ϩ)-strand] RNA virus genomes that function as signals for genome translation, transcription, and replication are potential sites for antiviral drug design. The fact that replication signals from evolutionarily divergent coronaviruses can be exchanged, for example, from the 3Ј untranslated region (UTR) of the (group 2a) bovine coronavirus (BCoV) to the (group 2a) mouse hepatitis coronavirus (MHV) (21)(22) or from the (group 2b) severe acute respiratory syndrome (SARS) coronavirus (SCoV) to MHV (15), would suggest that designed therapeutic agents against common signals might be broadly effective. Several studies have identified cis-replication structures in the 3Ј UTR of MHV, heretofore recognized as the group 2 type species, and in the 3Ј UTR of BCoV that are likely involved in the initial steps of genome translation and initiation of negativestrand [(Ϫ)-strand] synthesis. These elements, identified in either a helper virus-dependent defective interfering (DI) RNA of MHV or BCoV or by reverse genetics in the fulllength MHV genome, are the following: (i) the 3Ј poly(A) tail (26, 34), (ii) the 3Ј-terminal 55 nucleotides (nt) of the 3Ј UTR (14,23,26,42), and (iii) an upstream bulged stem-loop (SL) and associated hairpin pseudoknot (13,(21)(22)36). Curiously, an ϳ140-nt hypervariable region in the 3Ј UTR that comprises part of a 3Ј-proximal bulged stem-loop and harbors a coronavirus-universal octamer sequence (GGAAGAGC) is not required for MHV replication in cell culture but plays a role in MHV pathogenesis (14).cis-replication structures have also been identified in the 5Ј-proximal region of group 2 coronaviruses, but few studies have characterized their common signaling features. In BCoV, the very 5Ј end of the genome exhibits a puzzling hypervariability during virus replication in that 5Ј extensions of up to 9 nt and varying sequences within the first 14 nt are found, but the full significance of this in virus replication is unknown (19). The higher-order RNA structures within the BCoV 5Ј UTR were initially predicted by the Tinoco algorithm (35) and more recently by the mfold algorithm of Zuker (31, 41). They have been characterized as SLs I to IV (Fig. 1B) and were shown to be consistent with enzyme structure probing analyses (5-6, 32-33). More recent characterizations based on comparisons with MHV have shown SLI to be comprised of two smaller stem-loops, named SL1 and SL2 (Fig. 1B) (24)(25)(27)(28). In the context of the DI RNA in BCoV-infected cells, it was not feasible to determine whether SLI or SLII is a bona fide cisreplication element, since SLI resides within the 65-nt leader and SLII resides largely within the leader, and transfected DI RNAs containing an experimentally mutated leader rapidly acquired the leader sequence of the helper virus genome in a process known as "leader switching" (5-6, 29). SLII, furthermore, harbors the leader-associated transcription regulatory core sequence (UCUAAAC) responsible for the RNA-depen-* Corresponding author. Mailing addres...

Coronavirus cis-Acting RNA Elements

“…The SHAPE analyses also confirmed that the (weak) TRS-L-containing SL3 hairpin predicted previously by phylogenetic algorithms (Chen and Olsthoorn, 2010) is part of a single-stranded region, consistent with previous predictions that this region is weekly paired or unpaired (Liu et al, 2007;Madhugiri et al, 2014). Also several other RNA secondary structures identified by SHAPE analysis corresponded very well to the previous models of MHV-A59 RNA secondary structures proposed by Brian and coworkers (Guan et al, 2011(Guan et al, , 2012Yang et al, 2015). Furthermore, the study provides biochemical support for the presence of additional hairpin structures in the MHV 5 0 -terminal genome region, including SL5a (designated earlier as SL-IV), SL5b, SL5c, SL6, and SL7.…”

“…A 5 0 -terminal SL designated earlier as SL-IV that extends into the nsp1 coding sequence was described as an RNA element required for optimal MHV replication (Guan et al, 2011). The SHAPE analysis mentioned earlier suggests that SL5 contains three hairpin substructures, SL5a (previously designated as SL-IV), 5b, and 5c (Yang et al, 2015).…”

“…As outlined earlier, possible betacoronavirus SL5 substructures located within (or extending into) the nsp1-coding region (previously termed SLs IV, V, VI, and VII) have been characterized structurally and functionally using BCoV DI RNA and MHV reverse genetics systems (Brown et al, 2007;Guan et al, 2011Guan et al, , 2012Raman and Brian, 2005). In a BCoV-based DI RNA system, SL5A (previously designated as SL-IV) was revealed to be a cis-acting element essential for DI RNA replication (Brown et al, 2007).…”

MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA

“…These results suggest two possibi- lities: one is that the nsp1 protein carrying the R13A mutation is unable to bind to the 5′ UTR, and the other is that the R13A mutation disrupts cis-replication elements in the 5′ region of viral RNA. In fact, in other CoVs, the 5′ portion of the nsp1-coding region acts as a cisacting element, and the RNA structure of this region is critical for viral replication, as described previously (Brown et al, 2007;Chen and Olsthoorn, 2010;Guan et al, 2011). Therefore, as shown in Fig.…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…A possible SL5 equivalent in gammacoronaviruses was predicted to adopt a rod-like structure that lacks conserved loop sequences (Chen and Olsthoorn, 2010). As outlined above, possible betacoronavirus SL5 substructures located within (or extending into) the nsp1-coding region (termed SLs IV, V, VI, and VII) have been characterized structurally and functionally using BCoV DI RNA and MHV reverse genetics systems (Brown et al, 2007;Guan et al, 2011Guan et al, , 2012Raman and Brian, 2005).…”