Single point mutations in the pathogenicity domain of Potato spindle tuber viroid (PSTVd) can have a dramatic effect on disease expression, and only three substitutions are required for the spontaneous conversion of the type strain PSTVd-Intermediate to the rapidly replicating, highly pathogenic variant RG1 (Gruner et al., Virology209, 60–69, 1995). To identify available evolutionary pathways linking these two variants, we mutagenized five positions in an infectious cDNA copy of PSTVd-Intermediate and screened the resulting mixture of 768 sequences for neutral or near-neutral mutants. Numerical simulations based on the bioassay data indicate that the 23 variants recovered represent >80 % of all such sequences. RG1 was the only naturally occurring variant recovered, and the overall pattern of sequence changes observed indicates that PSTVd-Int occupies a comparatively steep peak within the fitness landscape.
AmariK.,
GomezG.,
MyrtaA.,
Di TerlizziB.,
PallásV.2001; The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule. J Gen Virol 82:953–962
AmbrósS.,
HernándezC.,
DesvignesJ. C.,
FloresR.1998; Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implication of the existence of constraints limiting the heterogeneity of viroid quasispecies. J Virol 72:7397–7406
EigenM.,
BiebricherC. K.1988; Sequence space and quasispecies distribution. In RNA Genetics vol. III, Variability of RNA Genomes pp 211–245 Edited by
DomingoE.,
HollandJ. J.,
AhlquistP.
Boca Raton, FL: CRC Press;
FarciP.,
ShimodaA.,
CoianaA.9 other authors2000; The outcome of acute hepatitis C predicted by evolution of the viral quasispecies. Science 288:339–344
HammondR. W.1992; Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site-directed mutagenesis. Virology 187:654–662
HuY.,
FeldsteinP. A.,
BottinoP. J.,
OwensR. A.1996; Role of the variable domain in modulating potato spindle tuber viroid replication. Virology 219:45–56
KeeseP.,
SymonsR. H.1985; Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc Natl Acad Sci U S A 82:4582–4586
KofalviS. A.,
MarcosJ. F.,
CañizaresM. C.,
PallásV.,
CandresseT.1997; Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J Gen Virol 78:3177–3186
LossP.,
SchmitzM.,
StegerG.,
RiesnerD.1991; Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J 10:719–727
LuckR.,
GrafS.,
StegerG.1999; ConStruct: a tool for thermodynamic controlled prediction of conserved secondary structure. Nucleic Acids Res 27:4208–4217
MatousekJ. A.,
PatzakJ.,
OrctováL.,
SchubertJ.,
VrbaL.,
StegerG.,
RiesnerD.2001; The variability of hop latent viroid as induced upon heat treatment. Virology 287:349–358
OwensR. A.1999; Viroids. In Encyclopedia of Virology
. , 2nd edn. pp 1928–1937 Edited by
GranoffA.,
WebsterR. G.
New York: Academic Press;
OwensR. A.,
ThompsonS. M.,
SieburthP. J.,
GarnseyS. M.2003; Limited sequence randomization: testing a strategy to produce improved citrus viroid dwarfing agents. In Proceedings 14th Conference International Organization of Citrus Virologists Riverside,CA: IOCV (in press)
PodleckisE. V.,
HammondR. W.,
HurttS. W.,
HadidiA.1993; Chemiluminescent detection of potato and pome fruit viroids by digoxygenin-labelled dot blot and tissue blot hybridization. J Virol Methods 43:147–158
PolivkaH.,
StaubU.,
GrossH. J.1996; Variation of viroid profiles in individual grapevine plants: novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I. J Gen Virol 77:155–161
QuF.,
HeinrichC. Loss P.,
StegerG.,
TienP.,
RiesnerD.1993; Multiple pathways of reversion in viroids for conservation of structural elements. EMBO J 12:2129–2139
Reidhaar-OlsonJ. F.,
SauerR. T.1988; Combinatorial cassette mutagenesis as a probe for the informational content of protein sequences. Science 241:53–57
SanoT.,
MimuraR.,
OhshimaK.2001; Phylogenetic analysis of hop and grapevine isolates of hop stunt viroid supports a grapevine origin for hop stunt disease. Virus Genes 22:53–59