An RNA Stem-Loop within the Bovine Coronavirus nsp1 Coding Region Is a cis -Acting Element in Defective Interfering RNA Replication

Abstract

ABSTRACT Higher-order cis -acting RNA replication structures have been identified in the 3′- and 5′-terminal untranslated regions (UTRs) of a bovine coronavirus (BCoV) defective interfering (DI) RNA. The UTRs are identical to those in the viral genome, since the 2.2-kb DI RNA is composed of only the two ends of the genome fused between an internal site within the 738-nucleotide (nt) 5′-most coding region (the nsp1, or p28, coding region) and a site just 4 nt upstream of the 3′-most open reading frame (ORF) (the N gene). The joined ends of the viral genome in the DI RNA create a single continuous 1,635-nt ORF, 288 nt of which come from the 738-nt nsp1 coding region. Here, we have analyzed features of the 5′-terminal 288-nt portion of the nsp1 coding region within the continuous ORF that are required for DI RNA replication. We observed that (i) the 5′-terminal 186 nt of the nsp1 coding region are necessary and sufficient for DI RNA replication, (ii) two Mfold -predicted stem-loops within the 186-nt sequence, named SLV (nt 239 to 310) and SLVI (nt 311 to 340), are supported by RNase structure probing and by nucleotide covariation among closely related group 2 coronaviruses, and (iii) SLVI is a required higher-order structure for DI RNA replication based on mutation analyses. The function of SLV has not been evaluated. We conclude that SLVI within the BCoV nsp1 coding region is a higher-order cis -replication element for DI RNA and postulate that it functions similarly in the viral genome.