RNA structures regulate norovirus life cycle and enable rational attenuation in vivo.
Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell chemical probing, revealing conserved motifs that regulate viral function. Targeted disruption of specific structural elements reduces viral replication in cell culture, modulates translation in cis, and decreases viral RNA levels in animal infection models. These findings enabled the rational design of a genetically stable, attenuated virus that elicits protective immunity and limits viral replication upon secondary challenge. Together, this work uncovers essential roles for RNA structure in norovirus biology and establishes a generalizable framework for RNA structure-guided design of antiviral vaccines and therapeutics.