Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast.
To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of approximately 30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.
Pogany
Judit
J
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA.
Nagy
Peter D
PD
D002457:Cell Extracts / Q000737:chemistry; D002474:Cell-Free System; D010957:Plasmids / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017864:Tombusvirus / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication
D002457:Cell Extracts; D012367:RNA, Viral