A compact viral IRES translates a downstream open reading frame.

Hepatitis C virus (HCV) and many other RNA viruses contain a Type IV internal ribosome entry site (IRES) in their 5' untranslated region (UTR). These IRES RNAs adopt a complex tertiary structure that interacts directly with the ribosome, enabling cap-independent translation initiation. Using bioinformatic methods to search viral genomes for more Type IV IRES RNAs, we discovered that megrivirus E (MeV-E) contains a putative Type IV IRES within its annotated 3' UTR. In addition to its unusual location downstream of the main coding region, the size of the MeV-E 3' IRES is substantially reduced compared to known Type IV IRESs. We confirmed the secondary structure of the MeV-E 3' IRES, determined its 3D structure in complex with the ribosome using cryoEM, and showed that the MeV-E 3' IRES initiates translation but at lower levels compared to the larger Type IV IRES in the MeV-E 5' UTR. We hypothesize that the absence of several domains in the MeV-E 3' IRES compared to other Type IV IRESs results in a loss of ribosomal protein interactions and a relative decrease in translation activity. This small Type IV IRES enables translation of a second open reading frame in the MeV-E genome, which likely encodes a transmembrane protein that is conserved in other megriviruses. We propose a model wherein MeV-E expresses lower levels of its downstream-encoded protein compared to those in the upstream coding region using a pared down IRES structure, demonstrating purposeful tuning of translation through RNA structural variation.