5’-Proximal cis-Acting RNA Signals for Coronavirus Genome Replication

RNA sequences and higher-order structures in the 5’ and 3’ untranslated regions (UTRs) of positive-strand RNA viruses are known to function as cis-acting elements for translation, replication, and transcription. In coronaviruses, these are best characterized in the group 2a bovine coronavirus (BCoV) and mouse hepatitis virus (MHV), yet their precise mechanistic features are largely undefined. Here, we use a reverse genetics system in MHV to exploit the ~30% nt sequence divergence between BCoV and MHV to establish structure/function relationships of 5’ UTR cis-replication elements. It had been previously shown that a precise replacement of the 391-nt MHV 3’ UTR with the 288-nt BCoV 3’ UTR yields wt-like MHV. Our attempts to replace the 209-nt MHV 5’ UTR with the 210-nt BCoV 5’ UTR, however, yielded a non-viable chimera. Therefore, a systematic analysis of individual 5’-terminal structures was made to identify compatible elements. By placing each of four putative cis-acting domains from the BCoV 5’ UTR into the MHV genome, we learned that (i) stem-loops (SLs) I & II and SLIII are functionally compatible, (ii) SLIV is compatible if it spans parts of the 5’ UTR and the nonstructural protein 1 (nsp1) cistron, thus identifying this part of ORF 1 as a component of the cis-replication signal, (iii) a relatively unstructured 32-nt region mapping between SLIII and SLIV defines a novel virus species-specific cis-replication element, (iv) spontaneous suppressor mutations within MHV SLI and nsp1 cistron compensated for growth defects arising from the BCoV 32-nt element in the MHV genome, (v) cross talk between the 32-nt element, SLI, and the nsp1 cistron appears essential for virus replication, (vi) the BCoV 5’ UTR and nsp1 cistron function together in the MHV genome to generate a wt-like MHV phenotype, and (vii) a functional 5’ UTR-nsp1 domain in group 2a coronaviruses cannot be substituted by the corresponding genomic element from the group 2b SARS-CoV. We postulate that the interaction between the 5’ UTR and nsp1 cistron (or possibly nsp1 protein) functions as a molecular switch between genome translation and ignition of negative-strand RNA synthesis.