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Replication of Tomato bushy stunt virus RNA in a plant in vitro system
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Abstract
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.