The mammalian orthoreovirus bicistronic M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require interaction of 5′–3′ untranslated regions

• The UTRs of MRV M3 mRNA do not possess cis-acting sequences/structures necessary for translation.
• The MRV M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism.
• The MRV M3 mRNA does not require the 3′ UTR or 5′–3′ UTRs interaction for translation initiation.
• The MRV M3 mRNA follows the translation initiation pattern in Kozak's context.
• The 3′ UTR of MRV M3 transcript does not affect the MRV viability or infection.

Mammalian orthoreovirus mRNAs possess short 5′ UTR, lack 3′ poly(A) tails, and may lack 5′ cap structures at late times post-infection. As such, the mechanisms by which these viral mRNAs recruit ribosomes remain completely unknown. Toward addressing this question, we used bicistronic MRV M3 mRNA to analyze the role of 5′ and 3′ UTRs during MRV protein synthesis. The 5′ UTR was found to be dispensable for translation initiation; however, reducing its length promoted increased downstream initiation. Modifying start site Kozak context altered the ratio of upstream to downstream initiation, whereas mutations in the 3′ UTR did not. Moreover, an M3 mRNA lacking a 3′ UTR was able to rescue MRV infection to WT levels in an siRNA trans-complementation assay. Together, these data allow us to propose a model in which the MRV M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require the viral mRNA 3′ UTR or 5′–3′ UTRs interaction.