Subcellular localization and role of Potato virus X (PVX) TGBp2 and TGBp3 in virus movement

Abstract

Scope and Method of Study: The objectives of this research were to explore the functions of Potato virus X (PVX) TGBp2 and TGBp3. The green fluorescent protein (GFP) was fused to the PVX TGBp2 or TGBp3 coding sequences and the fusions were inserted into PVX genomes or plasmids. Confocal microscopy was then used to study subcellular accumulation patterns of the fusion proteins in the presence and absence of virus infection. Mutations were introduced into the TGBp2 and TGBp3 coding sequences to identify domains controlling protein subcellular targeting. During the course of this thesis I established a system for studying PVX infection using BY-2 tobacco suspension cells. I also developed a fluorometric assay to measure turnover of GFP containing fusion proteins.

Findings and Conclusions: Novel TGBp2 induced vesicles were identified. These are ER-derived structures which are important for virus cell-to-cell movement. A central conserved amino acid sequence in PVX TGBp2 was shown to modulate vesicle morphology. Substitution of only a single amino acid was sufficient to change vesicle morphology, increase TGBp2 association with the ER, and inhibit PVX movement. A single mutation disrupting ER association of TGBp3, inhibited PVX cell-to-cell movement. Mutations near the C-terminus of TGBp3 delayed PVX cell-to-cell movement and inhibited vascular transport. These data indicate that the C-terminal cytosolic domain plays a role in virus movement that has not been previously described. Degradation of GFP:TGBp2 and TGBp3:GFP is stimulated by virus infection. These observations led to a model suggesting that viral induced ER stress and protein degradation may be linked to virus cell to-cell movement.