Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro

The tobacco etch potyviral protein, HC-Pro, is a multifunctional proteinase required for long-distance movement in plants and maintenance of genome replication at the single-cell level. It also functions in a counterdefensive capacity as a suppressor of posttranscriptional gene silencing (PTGS). To determine whether the requirements for HC-Pro during long distance movement and replication maintenance are due to the silencing suppressor function of the protein, a series of HC-Pro alanine scanning and other site-directed mutants were analyzed. Using a transient silencing suppression assay in Agrobacterium-injected leaf tissue, several suppression-defective mutants were identified. Each of six HC-Pro mutations, which were shown previously to confer long-distance movement and replication maintenance defects, conferred PTGS suppression defects. Interestingly, the genes encoding these defective HC-Pro derivatives were themselves susceptible targets of PTGS, resulting in low levels of mRNA and protein accumulation. Mutations that inactivated the proteinase domain active site had no effect on PTGS suppression function. The results are consistent with the hypothesis that the role of HC-Pro in long-distance movement and genome replication depends on PTGS suppression function and that this function is independent of HC-Pro proteolytic activity.