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Journal of Virology, September 2006, p. 8459-8468, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00545-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

Masaki Nishikiori,1,3 Koji Dohi,5,6 Masashi Mori,5,6 Tetsuo Meshi,2,6 Satoshi Naito,4 and Masayuki Ishikawa1,6*

Plant-Microbe Interactions Research Unit,1 Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305-8602,2 Graduate School of Agriculture,3 Graduate School of Life Science, Hokkaido University, Sapporo 060-8589,4 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi 921-8836,5 CREST, Japan Science and Technology Agency, Kawaguchi 322-0012, Japan6

Received 16 March 2006/ Accepted 4 June 2006

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

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

Journal of Virology, September 2006, p. 8459-8468, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00545-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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