Potato spindle tuber viroid (PSTVd) is believed to undergo a series of
specific structural transitions during replication. The variable doma
in of PSTVd is known to contain sequences that are important for repli
cation/accumulation as well as one of three ''premelting regions'' whi
ch control breakdown of the native structure in vitro. We have examine
d the structural and biological effects of five single and two double
nucleotide substitutions within premelting region 3 in an effort to is
olate temperature-sensitive mutations affecting PSTVd replication or p
athogenesis. None of these mutants replicated as rapidly as the wild t
ype, and a variety of spontaneous sequence changes were detected in th
eir progeny, Higher temperatures were able to partially overcome the i
nhibition of replication associated with a more stable secondary struc
ture, but no well-defined temperature-sensitive PSTVd mutants were ide
ntified. Selective pressures arising from the interaction of assay tem
perature and structural stability in vivo appear capable of moving PST
Vd populations between peaks of relatively high fitness. Depending on
the exact nature and location of the mutation, selection may occur at
the level of either the plus or the minus strand. (C) 1996 Academic Pr
ess, Inc.