Upstream ORFs as Regulators of Viral Replication

Upstream open reading frames (uORFs) are widespread regulatory elements located in the 5′-untranslated regions of eukaryotic and viral mRNAs. Once considered rare exceptions to the canonical scanning model, uORFs are now recognized as actively translated features present in approximately half of human transcripts, frequently initiating at non-canonical start codons. In eukaryotes, uORFs fine-tune protein synthesis in response to cellular stress by modulating ribosome reinitiation, as exemplified by the stress-dependent regulation of ATF4 and GCN4. In viral genomes, uORFs serve as compact, evolutionarily conserved modules that optimize protein stoichiometry, coordinate expression with the host cell state, and support persistent infection across diverse viral families through cis-regulation of translation. Beyond this, uORF-encoded peptides (uPeptides) can possess independent biological activities, influencing virulence, tissue tropism, and immune modulation. In this review, we systematize the available data on the dual functionality of uORFs as cis-regulatory elements of translation and as sources of bioactive uPeptides in viruses. Deciphering the full spectrum of uORF-mediated mechanisms may reveal novel layers of viral replication control and uncover potential targets for antiviral development, although, as discussed here, the available evidence for such applications is still largely limited to proof-of-principle mutagenesis.