Subcellular localization of the triple gene block proteins encoded by a foveavirus infecting grapevines

Grapevine rupestris stem pitting-associated virus (GRSPaV; Foveavirus; Flexiviridae) contains a positive-sense, ssRNA genome. GRSPaV occurs worldwide in grapes and is involved in the Rugose Wood disease complex. The GRSPaV genome contains the triple gene block (TGB), a genetic module present in several genera of plant RNA viruses. TGB encodes three proteins (TGBp1, TGBp2 and TGBp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. To reveal the subcellular localization of each TGB protein and to examine the impact that different fusion positions may have on the behavior of the native protein, we made a series of expression constructs and expressed the corresponding protein fusions in Nicotiana tabacum BY-2 cells and protoplasts. We demonstrated that TGBp1 had both a cytosolic and nuclear distribution. Two TGBp1 fusions (GFP fused at the N- or C-terminus) differ in subcellular distribution. Through the use of truncation mutants, we mapped TGBp1 regions responsible for the formation of two distinct types of aggregates. Sequence analyses predicted two and one transmembrane domains in TGBp2 and TGBp3, respectively. GFP fusions at either terminus of TGBp2 revealed identical localization to the ER network and ER-derived structures. In contrast, the two TGBp3 fusions to mRFP differed in localization. This is the first report on the subcellular localization of the viral proteins of a member of the Foveavirus genus.