The distribution of nucleotides across the genomes of viruses is non-random. The UpA dinucleotide (uracil ‘U’ bound to adenine ‘A’ by a phosphate bridge ‘p’) is under-represented in RNA viruses including Influenza A virus (IAV). This UpA suppression may aid IAV evasion of host immune responses, including UpA cleavage by type I interferon (IFN-I)-regulated endoribonucleases. We have added UpAs to the IAV genome in regions encoding the viral PB2 polymerase or nucleoprotein via synonymous mutations with the aim of reducing virus fitness while preserving wild-type (WT) epitopes. Compared to WT and control viruses, UpA-high (UpAH) mutants exhibit impaired replication in cells from several vertebrate host species with a marked depletion of UpAH transcripts, suggesting the presence of cellular UpA sensors which may alter RNA stability or transcription. UpAH IAV infection also triggers IFN-I production, however attenuation persists in cells defective for IFN-I production or signalling, indicating specific antivirals acting independently of the broader IFN-I response. RNase latent (RNaseL) and oligoadenylate-synthase 3 (OAS3) proteins have been previously implicated in UpA recognition and their role is being tested directly by infection of RNaseL/OAS3 knockout lines. RNA secondary structure modelling shows that the additional UpAs have predicted accessibility, facilitating potential recognition by UpA-responsive host factors. In order to identify novel UpA sensors, the UpAH viruses are being used in a human IFN-stimulated gene overexpression screen, targeting candidates like RNA-binding proteins and nucleases. Understanding UpA-driven IAV attenuation will reveal antiviral mechanisms that exploit dinucleotide composition and potentially guide vaccine design across RNA viruses.