Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts.
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.
Komoda
Keisuke
K
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Naito
Satoshi
S
Ishikawa
Masayuki
M
D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System; D016679:Genome, Viral; D059828:Plant Cells; D010944:Plants / Q000821:virology*; D011523:Protoplasts / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics* / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D011817:Rabbits; D014908:Triticum / Q000166:cytology; D014779:Virus Replication
D002457:Cell Extracts; D012367:RNA, Viral