The RNA-dependent RNA polymerase (RdRp) of Tomato bushy stunt virus (TBSV) contains an arginine- and proline-rich (RPR) motif. This motif functions as an RNA-binding domain and is essential for tombusvirus replication. A mutant carrying three arginine substitutions in this motif rendered the virus unable to replicate in Nicotiana benthamiana plants and protoplasts. When the replicase function was provided in trans, by expressing the TBSV RdRp in N. benthamiana plants, an infectious variant could be isolated. Sequence analysis showed that only the substituted glycine residue (position 216) had reverted to arginine; all other substitutions remained unchanged. This finding suggested that strong selection pressure is active to maintain necessary sequences of the viral RdRp and that the analysis of revertants may help to identify essential viral functions.
HorschR. B.,
FryJ. E.,
HoffmannN. L.,
EichholtzD.,
RogersS. G.,
FraleyR. T.1985; A simple and general method for transferring genes into plants. Science 227:1229–1231[CrossRef]
JablonskiS. A.,
LuoM.,
MorrowC. D.1991; Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol 65:4565–4572
KamerG.,
ArgosP.1984; Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res 12:7269–7282[CrossRef]
LongstaffM.,
BrignetiG.,
BoccardF.,
ChapmanS.,
BaulcombeD.1993; Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase. EMBO J 12:379–386
NagyP. D.,
PoganyJ.,
SimonA. E.2001; In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with Turnip crinkle virus
. Virology 288:315–324[CrossRef]
PanavieneZ.,
BakerJ. M.,
NagyP. D.2003; The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication. Virology 308:191–205[CrossRef]
PochO.,
SauvagetI.,
DelarueM.,
TordoN.1989; Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 8:3867–3874
RajendranK. S.,
NagyP. D.2003; Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. J Virol 77:9244–9258[CrossRef]
RubinoL.,
RussoM.1995; Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene. Virology 212:240–243[CrossRef]
WeaverS. C.,
DalgarnoL.,
FreyT. K.,
HuangH. V.,
KinneyR. M.,
RiceC. M.,
RoehrigJ. T.,
ShopeR. E.,
StraussE. G.2000; Togaviridae
. In Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses pp 879–889 Edited by
van RegenmortelM. H. V.,
FauquetD. H. L.,
BishopC. M.
San Diego: Academic Press;