Immunodetection, Expression Strategy and Complementation of Turnip Crinkle Virus p28 and p88 Replication Components

The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5′ end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs 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 detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a β-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5′-proximal open reading frame for any member of the family Tombusviridae.