Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5' end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5' end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs.
