Identification of the +1 ribosomal frameshifting site of LRV1-4 by mutational analysis.
Leishmania virus (LRV)1-4 has been reported to produce a fusion of ORF2 and ORF3 via a programmed +1 frameshift in the region where ORF2 and ORF3 overlap (Lee et al., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (nt. 2565-2823), frameshift region of LRV1-4, and the 71bp (nt. 2605-2678) sub-region where ORF2 and ORF3 overlap. We then predicted the frameshift site using a new computer program (Pseudoviewer), and finally identified the specific region associated with the mechanism of the LRV1-4's +1 frameshift by means of a mutational analysis based on the predicted structure of LRV1-4 RNA. The predicted structure was confirmed by biochemical analysis. In order to measure the frameshift efficiency, constructs that generate luciferase without a frameshift or with a +1 frameshift, were generated and in vitro transcription/translation analysis was performed. Measurements of the luciferase activity generated, showed that the frameshift efficiency was about 1% for both the 259bp (LRV1-4 259FS) and 71bp region (LRV1-4 71FS). Luciferase activity was strongly reduced in a mutant (LRV1-4 NH: nt. 2635-2670) with the entire hairpin deleted and in a mutant (LRV1-4 NUS: nt. 2644-2659) with the upper stem of the hairpin deleted. These results indicate that the frameshift site in LRV1-4's is in the 71bp region where ORF2 and ORF3 overlap, and that nt. 2644-2659 (the upward hairpin stem) play a key role in generating the +1 frameshift.
Kim
Se Na
SN
College of Pharmacy, Chung Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, Korea.
Choi
Jung Ho
JH
Park
Min Woo
MW
Jeong
Sun Joo
SJ
Han
Kyung Sook
KS
Kim
Hong Jin
HJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D018965:Frameshifting, Ribosomal; D017930:Genes, Reporter; D019199:Leishmaniavirus / Q000235:genetics*; D008156:Luciferases; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010957:Plasmids; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012984:Software; D014764:Viral Proteins / Q000235:genetics*
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014764:Viral Proteins; D008156:Luciferases