ABSTRACTCoxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5′ untranslated region (5′ UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5′ UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5′ UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5′ UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5′ UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5′ UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5′ UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA.IMPORTANCEHuman enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5′ untranslated region (5′ UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5′ UTR. We present RNA structure models that directly compare the 5′ UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.