Coronavirus-induced diseases in humans and animals are widespread and include acute respiratory disease, gastroenteritis, hepatitis, nephritis, and acute and chronic encephalitis. This proposal seeks to characterize five cis-acting higher-order RNA elements and one cis-acting process (translation) required for the replication of a coronavirus helper-dependent minireplicon (Dl RNA). These features are presumed requirements for the viral genome as well and are postulated to function in the formation of the RdRp replication complex. They also specifically address the enigma of how it is that coronavirus subgenomic mRNAs, synthesized on mRNA-length double-stranded intermediates and possessing termini identical to those on the genome (minimally 65 5' and 1633 3' nts), fail to replicate following transfection into helper virus-infected cells. The Dl RNA replicon is a 2.2 kb fusion product of the virus genomic termini and differs from mRNA 7 by only 421 nts of additional contiguous 5'-proximal sequence. It is postulated that sgmRNAs lack 3 of the necessary 5'-proximal signals for replication. The study has three specific aims: (1) To characterize the role of the phylogenetically-conserved 5'-proximal stem-loops Ill, IV, and V, including identification of the viral and cellular protein(s) that bind stem-loop III and the cellular protein(s) that binds stem-loop IV. (2) To characterize the cis-acting translation requirements for the (fused) partial la and entire N ORFs. (3) To characterize the 3' UTR cis-acting features of the phylogenetically conserved pseudoknot and its associated upstream bulged stem-loop, and the phylogenetically conserved octamer and its associated heptameric helix. Analyses will employ RNA structure probing, site-directed mutagenesis, mass-spectrometric identification of binding proteins, and assays for DI RNA minus-strand synthesis, replication and packaging.
