Abstract/Summary

Nidoviruses are enveloped, positive stranded RNA viruses that are among the most complex plus-strand RNA viruses known. My work has discovered the sequence of a new, highly divergent virus, tentatively named Abyssovirus, in a pool of sequenced intracellular RNA from a metazoan. Based on comparison of the genomic organization and replicase subunits, Abyssovirus appears to belong to a new family of nidoviruses. The aim of the present project was to validate the sequence data by studying the function of the predicted unusual translational stop-start signal located in the replicase gene, and to express predicted viral proteases and test their catalytic activity in Sf9 insect cells, E. coli and mammalian expression systems. The translational stop-start signal was found to allow ribosomes to readthrough the natural stop codon in the expression systems tested. Additionally, Abyssovirus Main Protease (MPro) showed evidence of cleavage of fragments from both ends of the expressed protease, as is typical for polyprotein-embedded Nidovirus proteases. These results suggest that the Abyssovirus RNA sequence encodes biologically functional proteins, and therefore that the RNA sequence likely does represent an extant virus with some Nidovirus-like features and an Abyssovirus-specific mechanism to translate the second half of the replicase open reading frame.