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J Virol, May 1998, p. 3965-3972, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Minimal Template Requirements for Initiation of Minus-Strand Synthesis In Vitro by the RNA-Dependent RNA Polymerase of Turnip Yellow Mosaic Virus

B. A. L. M. Deiman, A. K. Koenen, P. W. G. Verlaan, and C. W. A. Pleij*

Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden, The Netherlands

Received 30 July 1997/Accepted 19 January 1998

From mutational analysis of the 3'-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drastically, and deletion of only the 3'-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3' ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3' ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated Delta G value should be less than -10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

* Corresponding author. Mailing address: Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Phone: (31)715274769. Fax: (31)715274340. E-mail: c.pley{at}chem.leidenuniv.nl.

J Virol, May 1998, p. 3965-3972, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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