Replication of Tomato bushy stunt virus RNA in a plant in vitro system.
An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process.
Gursinsky
Torsten
T
Department Microbial Biotechnology, Institute of Biochemistry and Biotechnology, Faculty of Life Sciences (NatFakI), Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle/Saale, Germany.
Schulz
Beate
B
Behrens
Sven-Erik
SE
D002474:Cell-Free System; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014026:Nicotiana / Q000821:virology*; D017864:Tombusvirus / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase