The Bovine Coronavirus 2'-O-Methyltransferase Binds Cis-Acting Stem-Loop IV In the 5-Prime Untranslated Region Of The Viral Genome

The positive-stranded coronavirus genome, at 32 kilobases in length, is the largest known viral RNA genome, and internal cis-signaling elements directing its replication have been described only within the last ten years. The bovine coronavirus genome encodes 26 proteins in the region between the 5’-terminal 210-nt untranslated region and the 3’-terminal 298-nt untranslated region. Here, genes for 5 of the 26 proteins were cloned into bacterial expression plasmids for the long-term goals of characterizing enzymatic and RNA binding properties. These genes encode enzymes postulated to interact directly with the cis-acting RNA elements and carry out RNA synthesis, namely, the RNA-dependent RNA polymerase, the helicase, the exonuclease, the endonuclease, and the 2’-O-methyltransferase. For a detailed analysis, bacterially-expressed BCoV 2’-O-Methyltransferase was purified and (i) tested for enzymatic activity, which is presumably a 2’-O-methylation of 5’-terminal cap structures, and (ii) tested for its binding to terminal genomic regions known to contain cis-acting replication elements. Methyltransferase activity was not found, suggesting the proper conditions were not met or the proper template was not used, or perhaps, as with many viral enzymes made from a polyprotein precursor, it does not function as a unit-length molecule. Using the electrophoretic mobility shift assay, the 2’-O-Methyltransferase was found to bind cis-acting stem-loop IV in the 5’ untranslated region, but does not bind other cis-acting elements, including the region in gene 1 containing stem-loops V and VI or the 3’-proximal cis-acting bulged stem-loop and pseudoknot. The results of this study suggest that the putative bovine coronavirus 2’-O-Methyltransferase uses stem-loop IV as a binding site to carry out methyltransferase function(s) yet to be discovered.