In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. Their lengths range from 27,317 nt for HCoV-229E to 31,357 nt for the murine hepatitis virus-A59, establishing the coronavirus genome as the largest known among RNA viruses. The basic organization of the coronavirus genome is shared with other members of the Nidovirus order (the torovirus genus, also in the family Coronaviridae, and members of the family Arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mRNA synthesis (transcription) (an estimated 14-16 end products for coronaviruses) are encoded within the 5 0 -proximal two-thirds of the genome on gene 1 and the (mostly) structural proteins are encoded within the 3 0 -proximal one-third of the genome (8-9 genes for coronaviruses). Genes for the major structural proteins in all coronaviruses occur in the 5 0 to 3 0 order as S, E, M, and N. The precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. This chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (DI) RNA molecules], the minimum sequence requirements for autonomous replication of an RNA replicon, and the importance of gene order in genome replication. D

The genome of the porcine tanmisible gastroenteritis coronavirus is a plus-stand, polyadenylylated, infectious RNA molecule of "'20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-primng mensm to form a 3'-coterminal nested set. By using radiolabeled, strand-specifc, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leaderprimed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs.

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terininal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA

Secondary and tertiary structures in the 3′ untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3′ UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective interfering RNA genome. The pseudoknot is phylogenetically conserved among coronaviruses both in location and in shape but only partially in nucleotide sequence, and evolutionary covariation of bases to maintain G · U pairings indicates that it functions in the plus strand. RNase probing of synthetic transcripts provided additional evidence of its tertiary structure and also identified the possible existence of two conformational states. These results indicate that the 3′ UTR pseudoknot is involved in coronavirus RNA replication and lead us to postulate that it functions as a regulatory control element.