Turnip crinkle virus (TCV) (family Tombusviridae, genus Carmovirus) is a positive-sense RNA virus containing a 4054-base genome. Previous results indicated that insertion of Hairpin 4 (H4) into a TCV-associated satellite RNA enhanced replication 6-fold in vivo (Nagy, P., Pogany, J., Simon, A. E., 1999. EMBO J. 18:5653-5665). A detailed structural and functional analysis of H4 has now been performed to investigate its role in TCV replication. RNA structural probing of H4 in full-length TCV supported the sequence forming hairpin structures in both orientations in vitro. Deletion and mutational analyses determined that H4 is important for efficient accumulation of TCV in protoplasts, with a 98% reduction of genomic RNA levels when H4 was deleted. In vitro transcription using p88 [the TCV RNA-dependent RNA polymerase] demonstrated that H4 in its plus-sense orientation [H4(+)] caused a nearly 2-fold increase in RNA synthesis from a core hairpin promoter located on TCV plus-strands. H4 in its minus-sense orientation [H4(-)] stimulated RNA synthesis by 100-fold from a linear minus-strand promoter. Gel mobility shift assays indicated that p88 binds H4(+) and H4(-) with equal affinity, which was substantially greater than the binding affinity to the core promoters. These results support roles for H4(+) and H4(-) in TCV replication by enhancing syntheses of both strands through attracting the RdRp to the template.