Subcellular localization and role of potato virus X (PVX) TGBp2 in virus cell-to-cell movement

Potato virus X encodes three movement proteins (MP), TGBp1, TGBp2, and TGBp3. All three MPs, along with the viral coat protein (CP), are essential for virus cell-to-cell and systemic movement. TGBp2 and TGBp3 are putative membrane associated proteins. In this study we determined the importance of membrane association in protein and virus cell-to-cell movement. Many plant viruses move cell-to-cell via symplastic connections known as plasmodesmata (PD). The viruses must expand or gate the PD in order to move on to the adjacent cells. TGBp2 and TGBp3 cannot move through the PD in N. tabacum but can move cell-to-cell in transgenic tobacco expressing TGBp1. However the proteins can move independently in N. benthamiana, therefore the requirements of protein cell-to-cell movement is host specific. It was further determined that the TGBp2, and TGBp3 moved in source and not in the sink leaves of N. tabacum and N. benthamiana. The movement of the proteins, seem to depend also on the developmental stage of the leaf. Amino acid sequence analysis revealed that TGBp2 and TGBp3 have two and one putative transmembrane domains, respectively. Mutations introduced into the coding sequences encompassing the putative transmembrane domains within GFP:TGBp2 and GFP:TGBp3 disrupted membrane binding of those proteins. The GFP:TGBp2 and GFP:TGBp3 fusions were introduced into PVX genomes deficient for either TGBp2 or TGBp3. The targeting of TGBp2 and TGBp3 were monitored, along with the wild type PVX during virus infection using confocal microscopy. Both TGBp2 and TGBp3 were found to be associated with endoplasmic reticulum vesicles and inclusion bodies during the early stages of infection but became associated with the ER network during later stages of infection. The wild type PVX was cytosolic at all stages of virus movement. Further TGBp2 and TGBp3 mutations introduced in the transmembrane domains disrupted ER targeting of the PVX proteins. These mutations also inhibited PVX movement in N. tabacum and N. benthamiana when they were introduced in the virus genome revealing that ER association of the TGBp2 and TGBp3 is necessary for virus movement. Previous studies show that the requirements of protein movement may differ in the two hosts studied but the requirements of virus movement are the same.