The 3′-Terminal Structure Required for Replication of Barley Yellow Dwarf Virus RNA Contains an Embedded 3′ End

Abstract We determined the 3′-terminal primary and secondary structures required for replication of Barley yellow dwarf virus (BYDV) RNA in oat protoplasts. Computer predictions, nuclease probing, phylogenetic comparisons, and replication assays of specific mutants and chimeras revealed that the 3′-terminal 109 nucleotides (nt) form a structure with three to four stem-loops followed by a coaxially stacked helix incorporating the last four nt [(A/U)CCC]. Sequences upstream of the 109-nt region also contributed to RNA accumulation. The base-pairing but not the sequences or bulges in the stems were essential for replication, but any changes to the 3′-terminal helix destroyed replication. The two 3′-proximal tetraloops tolerated all changes, but the two 3′-distal tetraloops gave most efficient replication if they fit the GNRA consensus. A mutant lacking the 3′-proximal stem-loop produced elevated levels of less-than-full-length minus strands, and no (+) strand. We propose that a “pocket” structure is the origin of (−)-strand synthesis, which is negatively regulated by the inaccessible conformation of the 3′ terminus, thus favoring a high (+)/(−) ratio. This 3′ structure and the polymerase homologies suggest that genus Luteovirus is more closely related to the Tombusviridae family than to other Luteoviridae genera.