The 59 untranslated region (UTR) of the mouse hepatitis virus (MHV) genome contains cis-acting sequences necessary for transcription and replication. A consensus secondary structural model of the 59 140 nucleotides of the 59 UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem-loops, SL1, SL2, and SL4. NMR spectroscopy provides structural support for SL1 and SL2 in three group 2 CoVs, including MHV, BCoV, and HCoV-OC43. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5 base-pair stem, with some sequences containing an additional U 39 to U51; SL2 therefore possesses sequence features consistent with a U-turn-like conformation. The imino protons of U48 in the wild-type RNA, and G48 in the U48G SL2 mutant RNA, are significantly protected from exchange with solvent, consistent with a hydrogen bonding interaction critical to the hairpin loop architecture. SL2 is required for MHV replication; MHV genomes containing point substitutions predicted to perturb the SL2 structure (U48C, U48A) were not viable, while those that maintain the structure (U48G and U49A) were viable. The U48C MHV mutant supports both positive-and negative-sense genome-sized RNA synthesis, but fails to direct the synthesis of positive-or negative-sense subgenomic RNAs. These data support the existence of the SL2 in our models, and further suggest a critical role in coronavirus replication.

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Mouse hepatitis virus (MHV) is a positive-sense single-strand RNA virus of the order Nidovirales. MHV is a prototypical coronavirus; its genome is 32 kilobases in length and contains cis-acting elements responsible for viral mRNA transcription and genome replication (1,6,7,11,16,19,34). The genome also serves as an mRNA which contains two overlapping reading frames translated by an RNA frameshifting mechanism to produce a large polypeptide, which is autocatalytically proteolyzed into 16 different polypeptides, many of which contain or are predicted to contain a variety of enzymatic activities (31). These include RNA-dependent RNA polymerase, helicase, papain-like protease, picornavirus 3C-like protease, methyltransferase, and two different nuclease activities, all of which are likely required for viral replication. Coronavirus-infected cells contain five to eight subgenomic mRNAs, each of which is made up of two noncontiguous segments, an approximately 70-nucleotide (nt) leader RNA identical to the corresponding 5Ј end of the genome, which is joined to the downstream body of the mRNA (20, 28). Current thinking is that discontinuous transcription during negative-strand synthesis results in the production of a nested set of subgenomic negative-strand RNAs containing antileader RNAs that function as the sole templates for subgenomic mRNA synthesis (2, 26). Genome replication is thought to occur through the production of a negative-sense full-length RNA followed by synthesis of genomic RNA which is packaged and released as fully infectious virions.Most studies of coronavirus cis-acting replication signals have utilized defective interfering (DI) RNAs as model replicons due to the large size of coronavirus genomes. DIssE is a 2.2-kb, naturally occurring DI RNA which was isolated by serial passage of . DIssE is composed of three regions from the MHV-JHM genome. Region I is from nt 1 to 864, region II is derived from a 784-nt internal region 3.3 to 4 kb from the 5Ј end of the genome, and region III is derived from the 601 3Ј-terminal nucleotides (10). Studies of DIssE deletion mutants demonstrated that both the 5Ј-terminal 474 nt and the 3Ј-terminal 436 nt are necessary for replication, as is a 58-nt element derived from region II (1,11,19). Studies of various MHV-A59 DI RNAs have indicated that the requirement for the region II element is specific for MHV-JHM DI RNAs (11,25). Spagnolo and Hogue demonstrated that at least five A residues in the poly(A) tail are required for replication and poly(A) tail formation (29). Relatively few studies have attempted to dissect particular functions in genome replication for the various cis-acting sequences. Lin et al. demonstrated that DI RNAs in which the last 55 nt had been deleted were unable to serve as templates for negative-strand RNA

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