Translation Initiation in the SARS-CoV-2 5′ UTR: Canonical or Not?

The COVID-19 pandemic, caused by a novel coronavirus, SARS-CoV-2, has revealed many new aspects of coronavirus biology. Translation initiation of positive-sense, single-stranded RNA coronavirus is assumed to be cap-dependent due to the m7G cap at the 5’ end and the polyA tail at the 3’ end. However, structural analysis of the SARS-CoV-2 5’UTR suggests that an alternate translation mechanism could mediate translation initiation. To test the ability of non-canonical, cap-independent translation, we incorporated the SARS-CoV-2 5’ UTR into a plasmid containing dual-luciferase reporters. Using this bicistronic reporter gene system containing a Renilla luciferase and a Firefly luciferase, we measured both cap-dependent and cap-independent translation initiation. The Fuchs lab has generated several deletion mutants within the SARS-CoV2 5ʹ UTR. These include mutations of the upstream open reading frame (uORF). As an important control for these mutants, we generated the corresponding mono-luciferase constructs containing the SARS-CoV-2 5’ UTR and the firefly luciferase reporter. These mutant constructs were transfected into HeLa cells and luciferase activity was measured after 24 hours. These constructs act as an important control to substantiate the validity of IRES activity within the SARS-CoV-2 5’UTR. Interestingly, we have evidence that the SARS-CoV2 5ʹ UTR can indeed initiate translation internally. However, none of the translation initiation factors and other proteins, such as IRES transacting factors (ITAFs) have been identified. In the future, we will investigate the role of these proteins through modulation of their abundance in cells and determine how SARS-CoV2 translation from a the monocistronic and bicistronic reporters is affected. Understanding these proteins involved in the translation initiation pathways will provide insight into the critical step of translation initiation within the viral life cycle, furthering knowledge of how to potentially counteract infection.