Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase

Abstract

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.

Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo

“…Further support for the role of replicase binding to the intergenic region comes from previous work showing that a replicase binding domain is required for RNA synthesis from structured templates, whereas short, unstructured RNAs containing only an initiation site without binding domain are sufficient for RNA synthesis by the BMV replicase ( 38 ). A lack of RNA product from the structured template without binding domain suggested that inhibition occurred at or before initiation of RNA synthesis.…”

“…We show that apart from the 3′ end of RNA3, which contains the stem loop C replicase core promoter and initiation sequence, the replicase binding domain of the intergenic region is required for negative-strand synthesis. It has been postulated that the replicase scans an RNA template from the 5′ end in search of an initiation site and that intervening secondary structure, without a replicase binding domain, inhibits this scanning ( 38 ). Therefore, we suggest that binding of the replicase to the intergenic region guides the replicase to the 3′ end, bypassing natural secondary RNA structure.…”

The 3′-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

“…4C ). However, the involvement of 3′ UTR elements such as enhancers and repressors in the regulatory functions of replication has been suggested in RNA viruses [38] , [39] , [40] . Therefore, considering the replication efficiency of Δ55–45, Δ55–40 and Δ55–35 (44%, 102% and 78%, respectively), we speculate that nts −45 to −55 may serve as an enhancer because the deletion of these nts in mutant Δ55–45 leads to the modest inhibition of replication.…”

Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

“…Involvement of 5Ј-proximal sequences and structural elements in minusstrand synthesis has also been found for an increasing number of viruses (4,14,23,31,61,66,69). In addition, since plus strands cannot be templates for further translation during active replication (15), viral RNAs must contain the necessary information to switch between replication and translation as well as to support asymmetric synthesis of plus and minus strands, requirements that may include transcriptional enhancers (36,39,42,43) and repressors that function via RNA-RNA (39) or protein-RNA (11,73) interactions.…”