The poly(A) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during turnip mosaic virus infection

Abstract

Poly(A) binding protein 2 (PABP2) of Arabidopsis thaliana was previously shown to interact with VPg-Pro of turnip mosaic virus (TuMV) and may consequently play an important role during infection. Subcellular fractionation experiments revealed that PABP2 was predominantly a cytoplasmic soluble protein in healthy plants. However, in TuMV-infected plants, a subpopulation of PABP2 was membrane associated or was localized in the nucleus. Confocal microscopy experiments indicated that PABP2 was partially retargeted to the nucleolus in the presence of TuMV VPg-Pro. In addition, the membrane association of PABP2 during TuMV infection resulted from the internalization of the host protein in 6K-VPg-Pro-induced vesicles, as shown by a combination of confocal microscopy and sucrose gradient fractionation experiments. This redistribution of an important translation initiation factor to the nucleolus and to membrane structure likely underlies two important processes of the TuMV replication cycle.

FIG. 1.

Immunoblot analysis of soluble, membrane-associated, and nuclear proteins from healthy or TuMV-infected plants. B. perviridis plants were mock inoculated or infected with TuMV. (A) Twelve days after infection, total proteins (T) were extracted and soluble proteins (S) were separated from membrane-associated proteins (M) by centrifugation at 30,000 × g. Proteins were separated by SDS-PAGE and analyzed by Western blotting using a rabbit serum against PABP2. (B) Twelve days after infection, leaves were homogenized and centrifuged at 14,000 × g to separate the “soluble” fraction (S) from crude nuclei (N), which were further purified by Percoll gradient centrifugation. Proteins were separated by SDS-PAGE and analyzed by Western blotting using a rabbit serum against PABP2. The text on the left shows the electrophoretic migration positions of the indicated proteins.

FIG. 2.

Subcellular localizations of PABP2, eIF(iso)4E, and VPg-Pro. N. benthamiana leaves were infiltrated with A. tumefaciens, and expression of fluorescent proteins was visualized by confocal microscopy 4 to 5 days later. A. tumefaciens suspensions contained binary Ti plasmids encoding PABP2-GFP and ER-DsRed2 (A), PABP2-GFP and VPg-Pro-DsRed2 (B and C), VPgPro-DsRed2 and Atfib2-GFP (D), eIF(iso)4E-DsRed2 and GFP-ER (E), eIF(iso)4E-DsRed2 and VPg-Pro-GFP (F and G), and PABP2-mCherry, ntGFP-eIF(iso)4E, and VPg-Pro-ctGFP (H and I). Left panels show fluorescence emitted by the red channel only, while middle panels show fluorescence emitted by the green channel only, and right panels show the merge between the red and green channels. C, G, and I are close-up views of the squares depicted in B, F, and H, respectively. Bar, 10 μm.

FIG. 3.

VPg-Pro interaction with PABP2 and eIF(iso)4E of A. thaliana demonstrated by ELISA-based binding assay. (A) Wells were coated with 1.0 μg purified VPg-Pro and then incubated with increasing concentrations of purified T7-tagged eIF(iso)4E with (▪) or without (⧫) His-tailed PAPB2 (1.6 μg). Retention of the complex was detected using anti-T7-tagged (filled symbols) or anti-His (open symbols) antibodies. (B) Wells were coated with 1.0 μg purified VPg-Pro and then incubated with increasing concentrations of purified His-tailed PAPB2 with (▪) or without (⧫) T7-tagged eIF(iso)4E (1.6 μg). Retention of the complex was detected using anti-T7-tagged (filled symbols) or anti-His (open symbols) antibodies. Values are means of four replicates from a typical experiment. Error bars represent standard deviations. O.D., optical density.

FIG. 4.

Subcellular localization of eIF(iso)4E, PABP2, and 6K-VPg-Pro. N. benthamiana leaves were infiltrated with A. tumefaciens, and expression of fluorescent proteins was visualized by confocal microscopy 4 to 5 days later. A. tumefaciens suspensions contained binary Ti plasmids encoding PABP2-mCherry and 6K-VPg-Pro-GFP (A and B) or PABP2-mCherry, ntGFP-eIF(iso)4E, and 6K-VPg-Pro-ctGFP GFP (C and D). Left panels show fluorescence emitted by the red channel only, while middle panels show fluorescence emitted by the green channel only, and right panels show the merge between the red and green channels. B and D are close-up views of the squares depicted in A and C, respectively. Bar, 10 μm.

FIG. 5.

Membrane flotation assays. P30 fractions were used. Fractions were collected from the step sucrose gradient, and proteins present in each collected fraction were separated by SDS-PAGE (12%) and immunodetected with the anti-BiP (A), VPg-Pro (B), and PABP2 (C) antibodies.

FIG. 6.

Detection of proteins in membrane fractions following centrifugation in a sucrose gradient. The P30 fraction was prepared and centrifuged on 20 to 45% sucrose density gradients. The direction of sedimentation was from right to left, with fraction 15 representing the top of the gradient. Fractions were collected, proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose, and the immunoblot was analyzed using anti-VPg-Pro (A), anti-RdRp (B), or anti-PABP2 (C) serum.

FIG. 7.

Copurification of 6K-VPg-Pro/VPg-Pro and PABP2 by metal chelation chromatography. Membrane fractions were solubilized by the addition of 40 mM octyl-β-glucopyranoside loaded on to a column containing 0.4 ml nickel-agarose resin. Proteins were eluted with 100 mM imidazole. Total (T), membrane-associated (M), and eluted (E) proteins from mock-inoculated and TuMV-infected B. perviridis plants were separated by SDS-PAGE and electroblotted onto nitrocellulose. The membrane was probed using anti-VPg-Pro (A) and anti-PABP2 (B) sera.

FIG. 8.

Biochemical treatments of membrane-enriched fractions derived from TuMV-infected B. perviridis. Membrane-enriched (P30) fractions were treated with 1 M KCl or 0.1 M Na₂CO₃ (pH 10.5) for 30 min at 4°C. After separation of membrane-bound (P) and soluble (S) proteins, the presence of BiP (A), VPg-Pro (B), RdRp (C), and PABP2 (D) in these fractions was revealed by immunoblotting with the respective serum.