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Journal of Virology, March 2007, p. 2584-2591, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.01921-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation{triangledown}

Keisuke Komoda,1,2 Natsuki Mawatari,1,3 Yuka Hagiwara-Komoda,1,4 Satoshi Naito,2 and Masayuki Ishikawa1,4*

Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan,1 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan,2 Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan,3 CREST, Japan Science and Technology Agency, Kawaguchi 322-0012, Japan4

Received 4 September 2006/ Accepted 5 November 2006

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.

* Corresponding author. Mailing address: Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. Phone and fax: 81-29-838-7009. E-mail: ishika32{at}affrc.go.jp.

{triangledown} Published ahead of print on 15 November 2006.

Journal of Virology, March 2007, p. 2584-2591, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.01921-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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