The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis.
A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.
Chen
I-Hsuan
IH
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
Lin
Jen-Wen
JW
Chen
Yi-Jing
YJ
Wang
Zi-Chao
ZC
Liang
Li-Fang
LF
Meng
Menghsiao
M
Hsu
Yau-Heiu
YH
Tsai
Ching-Hsiu
CH
D000227:Adenine Nucleotides / Q000378:metabolism; D031723:Bambusa / Q000187:drug effects / Q000528:radiation effects / Q000821:virology*; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016679:Genome, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000528:radiation effects; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D018515:Plant Leaves / Q000187:drug effects / Q000528:radiation effects / Q000821:virology; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic; D014026:Nicotiana / Q000187:drug effects / Q000528:radiation effects / Q000821:virology; D014466:Ultraviolet Rays; D014529:Uridine / Q000378:metabolism
D000227:Adenine Nucleotides; D003432:Cross-Linking Reagents; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D014529:Uridine
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