RNA structure analysis of alphacoronavirus terminal genome regions

Abstract

Coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. Interactions of the viral replicase complex with cis-acting RNA elements located in the 5' and 3'-terminal genome regions ensure the specific replication of viral RNA. Over the past years, boundaries and structures of cis-acting RNA elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. Here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and RNA structure probing studies to identify and characterize putative cis-acting RNA elements in alphacoronaviruses. The study suggests significant RNA structure conservation among members of the genus Alphacoronavirus but also across genus boundaries. Overall, the conservation pattern identified for 5' and 3'-terminal RNA structural elements in the genomes of alpha- and betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the Coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting RNA elements.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Similar to that of other positive-strand RNA viruses, coronavirus RNA synthesis requires the specific recognition of cis -acting RNA elements, which are mainly located in the highly structured 5′ and 3′ untranslated regions (UTRs), although they may also extend into the adjacent coding sequences (9, 100, 101). Such cis -acting RNA elements in the 5′ end of the coronavirus genome were first studied in BCoV using defective interfering RNAs.…”

“…More recently, these studies have been extended to other betacoronaviruses and, to a lesser extent, to alpha- and gammacoronaviruses. These RNA elements mainly consist of stem-loop (SL) structures that present a varying degree of conservation among different coronaviruses (9, 101–103). In order to consolidate the information from various publications, we adopt a uniform nomenclature for the 5′ cis -acting RNA elements (Figure 4) based on that used by the Leibowitz, Giedroc, and Olsthoorn laboratories (102, 103).…”

“…In order to consolidate the information from various publications, we adopt a uniform nomenclature for the 5′ cis -acting RNA elements (Figure 4) based on that used by the Leibowitz, Giedroc, and Olsthoorn laboratories (102, 103). SL1 and SL2 are conserved in all coronaviruses (101, 102). SL1 adopts a bipartite structure (104), and SL2 presents a highly conserved loop sequence that adopts a YNMG- or CUYG-type tetraloop conformation (105, 106).…”

“…SL3, which contains the leader CS, is involved in discontinuous transcription as a receptor for nascent negative-strand RNA. Downstream of the leader CS is SL4, a long hairpin that is structurally conserved in all coronavirus genera (101, 102). SL4 is essential for replication of BCoV defective interfering RNA (107), and it was proposed to function as a spacer element that controls the orientation of upstream SLs driving sgmRNA synthesis (108).…”

“…Finally, a higher-order RNA structure (SL5) that extends into ORF1a seems to be conserved within specific coronavirus genera (102). In alphacoronaviruses, SL5 may be further subdivided into three hairpins (SL5a, SL5b, and SL5c), which are partially conserved in betacoronaviruses (101). In gammacoronaviruses, a possible SL5 is predicted to adopt a rod-like structure (102).…”