Abstract

Since the discovery of a short, essential potyviral ORF , pipo, in the -1 reading frame relative to the polyprotein ORF (Chung et al. 2008 PNAS 105 , 5897), we have investigated the function of the pipo-encoded protein, PIPO . pipo overlaps with the P3 coding region and is translated by ribosomal frameshifting as a fusion to the N terminus of P3; antibodies that specifically detect either PIPO or N-terminus of P3 both recognize a 25 kDa protein in Turnip mosaic virus (TuMV)-infected cells. We call this trans-frame fusion protein P3N -PIPO. To determine its function, we identified host proteins with which it interacts in a yeast two-hybrid screen of anArabidopsis thaliana cDNA expression library. One of the interacting proteins is a plasma membrane-associated protein, we call host factor 3 (AtHF3). The interaction of P3N -PIPO with AtHF3 was validated in planta by co-immunoprecipitation of both proteins, which had been co-expressed by agroinfiltration, and also by bimolecular fluorescence complementation assay. To determine the requirement, if any, for AtHF3 inTuMV infection, expression of the AtHF3 ortholog in N. benthamiana (NbHF3) was knocked down by VIGS , and plants were then challenged with TuMV engineered to express GFP . Based on qRT-PCR analysis, knockdown of HF3 expression reduced accumulation of viral RNA in inoculated leaves. Also, appearance of disease symptoms and green fluorescence were reduced dramatically in inoculated leaves and delayed in upper leaves. Thus HF3 is required for efficient virus infection, and it may play a role in virus movement. Indeed, bombardment of leaves with a plasmid expressing P3N -PIPO-GFP fusion protein revealed that P3N -PIPO facilitates its own cell-to-cell movement. These observations are consistent with others’ results that suggested a movement role for P3N -PIPO. In summary, HF3 is a host protein not known previously to participate in virus infection that may contribute to potyviral intercellular trafficking.