Resurrection of a viral internal ribosome entry site from a 700 year old ancient Northwest Territories cripavirus

Abstract

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. Structural prediction of the aNCV IGR sequence generated a secondary structure similar to contemporary IGR IRES structures. There are, however, subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IGR IRES could bind to purified salt-washed human ribosomes and compete with a prototypical IGR IRES for ribosomes. Toeprinting analysis using primer extension pinpointed the putative start site of the aNCV IGR at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR IRES can direct internal ribosome entry in vitro in a manner dependent on the integrity of the PKI domain. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework in the importance of this viral translational mechanism. IMPORTANCEInternal ribosome entry sites are RNA structures that are used by some positive-sense monopartite RNA viruses to drive viral protein synthesis. The origin of internal ribosome entry sites is not known. Using biochemical approaches, we demonstrate that an RNA structure from an ancient viral genome that was discovered from a 700-year-old caribou feces trapped in subarctic ice is functionally similar to modern internal ribosome entry sites. We resurrect this ancient RNA mechanism by demonstrating that it can support virus infection in a contemporary virus clone, thus providing insights into the origin and evolution of this viral strategy.