A cis-replication element functions in both orientations to enhance replication of Turnip crinkle virus.
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.
Sun
Xiaoping
X
Department of Cell Biology and Molecular Genetics, Microbiology Building, University of Maryland College Park, College Park, MD 20742, USA.
Simon
Anne E
AE
D001483:Base Sequence; D029688:Brassica napus / Q000821:virology*; D019184:Carmovirus / Q000235:genetics / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D013329:Structure-Activity Relationship; D014779:Virus Replication
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase