The structural and phylogenetic profile of the 3′ terminus of coxsackievirus B3 negative strand

• The structure of the 3′-terminal region of CV-B3 minus strand was determined.
• The 3′-terminal region of 787 nucleotides folds independently of upstream sequence.
• Several structurally conserved motifs are identified in the analyzed viral region.
• A cloverleaf structural motif is present at the 3′-end of the CV-B3 negative strand.
• The conserved motifs of the 3′-terminal region may be crucial in viral replication.

In the replication process of RNA(+) viruses both the positive-strand template and the newly synthesized negative strand appear in a double-stranded form, RF. It has been shown for poliovirus that prior to the initiation of positive-strand synthesis, the 5′-terminus of the positive strand must adopt a cloverleaf structure. When that happens, the 3′-terminal region of the negative strand is released from the RF form and is able to form into its own defined structure. In order to determine the secondary structure of this region, a comprehensive approach consisting of experimental mapping methods, phylogenetic analysis and computer predictions was applied. Here we propose the first structural model of the 3′-terminal region of the coxsackievirus B3 (CV-B3) negative strand, approximately 450 nucleotides in length. The region folds into three highly defined structural domains, I′–III′. The most 3′-terminal part of this region is domain I′, which folds into a cloverleaf structure similar to that found in the viral RNA strand of positive-polarity. Remarkably, this motif is conserved among all analyzed viral isolates of CV-B3 despite the observed sequence diversity. Several other conserved structural motifs within the 3′-terminal region of the viral negative strand were also identified. The structure of this region may be crucial for the replication complex assembly.