An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs.
Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.
Ray
Debashish
D
Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3.
White
K Andrew
KA
D001483:Base Sequence; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D017864:Tombusvirus / Q000235:genetics*
D012367:RNA, Viral