Dependence of Coronavirus RNA Replication on an NH2-Terminal Partial Nonstructural Protein 1 in cis

Genomes of positive ()-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5=-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5 = untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open read-ing frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a re-cently discovered long-range RNA-RNA base-paired structure between the 5 = untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the () strand.We postu-late that the higher-order structure promotes ()-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH2-proximalWAPEFPWMdomain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and ex-plain, in part, why coronavirus sgmRNAs fail to replicate