Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in Planta

Abstract

The RNA genome of Turnip mosaic virus is covalently linked at its 5' end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.

FIG. 1.

Immunoblot analysis of soluble and membrane-associated proteins from healthy or TuMV-infected plants. B. perviridis plants were mock inoculated or infected with TuMV. Twelve days later, total proteins (T) were extracted and soluble proteins (S) were separated from membrane-associated proteins (M) by centrifugation at 27,000 × g. Proteins were separated by SDS-PAGE and analyzed by Western blotting using a rabbit serum against eIF(iso)4E (A) or VPgPro (B). The text on the right shows the electrophoretic migration positions of the indicated proteins.

FIG. 2.

Expression of GFP fusions in N. benthamiana. Leaves were infiltrated with A. tumefaciens; 4 days later, total proteins were extracted, separated by SDS-PAGE, and analyzed by Western blotting using a rabbit serum against VPgPro. A. tumefaciens suspensions contained binary Ti plasmids encoding VPg-Pro-GFP (lane 1), VPg-Pro-DsRed2 (lane 2), VPg-Pro-ctGFP (lane 3), 6K-VPg-Pro-GFP (lane 4), 6K-VPg-Pro-ctGFP (lane 5), and GFP (lane 7). Proteins from TuMV-infected B. perviridis were loaded in lane 6.

FIG. 3.

Subcellular localizations of eIF(iso)4E and VPg precursors. N. benthamiana leaves were infiltrated with A. tumefaciens, and expression of fluorescent proteins was visualized by confocal microscopy 4 days later. A. tumefaciens suspensions contained binary Ti plasmids encoding eIF(iso)4E-GFP and ER-DsRed2 (A to D), GFP (E), VPg-Pro-DsRed2 and GFP-ER (F), VPg-Pro-GFP and ER-DsRed2 (G to I), VPg-Pro-GFP and DsRed2 (J to L), and 6K-VPg-Pro-GFP and ER-DsRed2 (M to O). Panel M is a “maximum-intensity Z projection” of 50 1-μm slices stacked on top of each other. Panels A, E, G, and J show fluorescence emitted by the green channel only; panels B, H, K, and O show fluorescence emitted by the red channel only. Panel D is a close-up view of the rectangle depicted in panel C. The arrow in panel O shows an outgrowth of ER. Bar, 15 μm.

FIG. 4.

Subcellular localizations of the interaction between eIF(iso)4E and VPg precursors. N. benthamiana leaves were infiltrated with A. tumefaciens, and expression of fluorescent proteins was visualized by confocal microscopy 4 days later. A. tumefaciens suspensions contained binary Ti plasmids encoding VPg-Pro-ctGFP, ntGFP-eIF(iso)4E, and ER-DsRed2 (A to D); ctGFP and ntGFP-eIF(iso)4E (E); or 6K-VPg-Pro-ctGFP, ntGFP-eIF(iso)4E, and ER-DsRed2 (F to I). Panel F is a “maximum-intensity Z projection” of 13 1-μm slices stacked on top of each other. Panels B to D and I are close-up views of the rectangles depicted in panels A and H, respectively. Panel B shows fluorescence emitted by the green channel only; panels C, H, and I show fluorescence emitted by the red channel only. Bar, 15 μm.

FIG. 5.

Immunoblot analysis of nuclear and postnuclear fraction proteins from healthy or TuMV-infected plants. B. perviridis plants were mock inoculated or infected with TuMV. Twelve days later, leaves were homogenized and centrifuged at 14,000 × g to separate the “soluble” fraction (S) from crude nuclei, which were further purified by Percoll gradient centrifugation (N). Proteins were separated by SDS-PAGE and analyzed by Western blotting using a rabbit serum against Bip (A), VPg-Pro (B), or eIF(iso)4E (C). The text on the right shows the electrophoretic migration positions of the indicated proteins.