Molecular analysis of the role of IRES stem-loop V in replicative capacities and translation efficiencies of Coxsackievirus B3 mutants

Abstract

Coxsackievirus B3 (CVB3) is a principal viral cause of acute myocarditis in humans and has been implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, it has been demonstrated elsewhere that, for several wild-type CB3 strains, the primary molecular determinant of cardiovirulence phenotype localizes to the 5′ nontranslated region (5′NTR) and capsid. Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA–RNA and RNA–protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on one most frequently occurring family of tetraloop sequences, namely, the GNRA sequence, especially the GNAA sequence conserved in all enteroviruses. Here in this study, through construction of CVB3 chimeric mutants, the predicted stem loop (SL) V within the 5′NTR has been identified as important in determining viral cardiovirulence. Replication assays in HeLa cell monolayers revealed that wild-type CVB3 virus and two of the six mutants constructed here grow efficiently, whereas other mutant viruses replicate poorly. Furthermore, the in vitro translation products from these mutants and wild-type CVB3, demonstrated that the two mutants who replicate efficiently, translated at relatively equivalent amount than the wild-type. However, other mutants demonstrated a low efficiency in their production of protein when translated in a Rabbit Reticulocytes Lysats.