Genetic characterization of the coxsackievirus B2 3' untranslated region

The secondary structure of the 3' untranslated region (3'UTR) of picornaviruses is thought to be important for the initiation of negative-strand RNA synthesis. In this study, genetic and biological analyses of the 3' terminus of coxsackievirus B2 (CVB2), which differs from other enteroviruses due to the presence of five additional nucleotides prior to the poly(A) tail, is reported. The importance of this extension was investigated using a 3'UTR mutant lacking the five nucleotides prior to the poly(A) tail and containing two point mutations. The predicted secondary structure within the 3'UTR of this mutant was less energetically favourable compared with that of the wild-type (wt) genotype. This mutant clone was transfected into green monkey kidney cells in four parallel experiments and propagated for multiple passages, enabling the virus to establish a stable revertant genotype. Genetic analysis of the virus progeny from these different passages revealed two major types of revertant. Both types showed wt-like growth properties and more stable and wt-like predicted secondary structures than the parent mutant clone. The first type of revertant neutralized the introduced point mutation with a compensatory second-site mutation, whereas the second type of revertant partly compensated for the deletion of the five proximal nucleotides by the insertion of nucleotides that matched the wt sequence. Therefore, the extended 3' end of CVB2 may be considered to be a stabilizing sequence for RNA secondary structure and an important feature for the virus.