Ka. White et al., IMMUNODETECTION, EXPRESSION STRATEGY AND COMPLEMENTATION OF TURNIP CRINKLE VIRUS P-28 AND P88 REPLICATION COMPONENTS, Virology, 211(2), 1995, pp. 525-534
The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its
5' end a 28-kDa protein of unspecified function. Readthrough suppress
ion of the p28 stop codon allows for the production of an 88-kDa produ
ct which is required for genome replication. Immunological analysis of
the expression of p28 and pas demonstrated that: (i) the genome direc
ts the synthesis of polypeptides of approximately 28 and 88 kDa, (ii)
the 88-kDa protein is immunologically related to p28, consistent with
p88 being a readthrough product, and (iii) p28, but not p88, is detect
able in vivo. An in vivo assay, in which readthrough is linked to the
expression of a beta-glucuronidase reporter gene, showed that readthro
ugh of the p28 amber stop codon occurs with an efficiency of approxima
tely 1%. A similar efficiency of readthrough was observed when an alte
red context from the nonviable TCV mutant, mA2, containing a disrupted
secondary structure (FfFa) spanning the p28 termination codon, was te
sted. This result suggests that the defective phenotype of mA2 is like
ly not linked to an alteration in readthrough efficiency. Additional s
tudies demonstrated that complementation occurs in coinoculations with
two nonviable TCV mutants, RT and APA, which are unable to express ei
ther p28 or p88, respectively. This result verifies that p28 is essent
ial for TCV genome replication and provides the first definitive evide
nce for the role of a 5'-proximal open reading frame for any member of
the family Tombusviridae. (C) 1995 Academic Press, Inc.