Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

doi:10.1073/pnas.0307131101

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

^*Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan; and ^†Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 322-0012, Japan

Edited by George Bruening, University of California, Davis, CA, and approved December 22, 2003 (received for review November 4, 2003)

Abstract

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: ishikawa{at}abs.agr.hokudai.ac.jp.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ToMV, tomato mosaic virus; BMV, brome mosaic virus; TCV, turnip crinkle virus; RRL, rabbit reticulocyte lysate; WGE, wheat germ extract; RF, replicative form.