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Journal of Virology, July 2005, p. 9046-9053, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.9046-9053.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

Jen-Wen Lin,{dagger} Hsiao-Ning Chiu,{dagger} I-Hsuan Chen, Tzu-Chi Chen, Yau-Heiu Hsu, and Ching-Hsiu Tsai*

Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan

Received 6 December 2004/ Accepted 13 March 2005

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3'-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3' terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/{Delta}5, with a deletion of the 3'-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/{Delta}16 and Ba-77/{Delta}31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3' end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

* Corresponding author. Mailing address: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan. Phone: (886) 4-22840451. Fax: (886) 4-22860260. E-mail: chtsai1{at}dragon.nchu.edu.tw.

{dagger} Both authors contributed equally to this work.

Journal of Virology, July 2005, p. 9046-9053, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.9046-9053.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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