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. 2008 Dec;82(24):12252-64.
doi: 10.1128/JVI.01329-08. Epub 2008 Oct 8.

Biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a COPI- and COPII-dependent manner

Taiyun Wei  1 , Aiming Wang
Affiliations

Biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a COPI- and COPII-dependent manner

Taiyun Wei et al. J Virol. 2008 Dec.

Abstract

Single-stranded positive-sense RNA viruses induce the biogenesis of cytoplasmic membranous vesicles, where viral replication takes place. However, the mechanism underlying this characteristic vesicular proliferation remains poorly understood. Previously, a 6-kDa potyvirus membrane protein (6K) was shown to interact with the endoplasmic reticulum (ER) and to induce the formation of the membranous vesicles. In this study, the involvement of the early secretory pathway in the formation of the 6K-induced vesicles was investigated in planta. By means of live-cell imaging, it was found that the 6K protein was predominantly colocalized with Sar1, Sec23, and Sec24, which are known markers of ER exit sites (ERES). The localization of 6K at ERES was prevented by the coexpression of a dominant-negative mutant of Sar1 that disables the COPII activity or by the coexpression of a mutant of Arf1 that disrupts the COPI complex. The secretion of a soluble secretory marker targeting the apoplast was arrested at the level of the ER in cells overexpressing 6K or infected by a potyvirus. This blockage of protein trafficking out of the ER by 6K and the distribution of 6K toward the ERES may account for the aggregation of the 6K-bound vesicles. Finally, virus infection was reduced when the accumulation of 6K at ERES was inhibited by impairing either the COPI or COPII complex. Taken together, these results imply that the cellular COPI and COPII coating machineries are involved in the biogenesis of the potyvirus 6K vesicles at the ERES for viral-genome replication.

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Figures

FIG. 1.
FIG. 1.
6K is localized at the ERES and at additional ringlike structures on the ER membrane in N. benthamiana leaves. (A) Confocal images of cells expressing 6K-CFP (I) and VPg-YFP (II). Panels III and IV show a bright-field image and merged images, respectively. (B) Confocal images of cells expressing YFP-HDEL alone (I) or in combination with 6K-CFP (II to IV). The arrows indicate colocalization of punctate structures of 6K with cortical aggregates of the ER membrane. The arrowheads indicate colocalization of ringlike structures of 6K with cortical aggregates of the ER membrane. (C) Confocal images of cells expressing YFP-Sec24 alone (I) or in combination with 6K-CFP (II to IV). The arrows indicate colocalization of punctate structures of 6K-CFP with YFP-Sec24-labeled ERES. The arrowheads indicate that the ringlike structures did not colocalize with the ERES labeled by YFP-Sec24. (D) Confocal images of cells expressing YFP-Sec23 alone (I) or in combination with 6K-CFP (II to IV). The arrows indicate colocalization of punctate structures of 6K-CFP with YFP-Sec23-labeled ERES. The arrowheads indicate the ringlike structure that did not colocalize with the ERES staining by YFP-Sec23. Bars, 9 μm.
FIG. 2.
FIG. 2.
Coalignment of 6K vesicles with microfilaments. (A) Expression of 6K-CFP and mTalin-YFP in an N. benthamiana leaf epidermal cell after agroinfiltration. The arrows indicate coalignment of microfilament markers with 6K-CFP. (B) LatB at 25 μM concentration was infiltrated into N. benthamiana leaves coexpressing 6K-CFP and mTalin-YFP. At 2 h postinfiltration, the infiltrated area was observed by confocal laser scanning microscopy. The 6K-CFP vesicles still formed after treatment with Lat B but did not always coalign with collapsed microfilaments (B). Bars, 9 μm.
FIG. 3.
FIG. 3.
Golgi stacks and 6K-YFP punctate structures move together. (A) Confocal images of N. benthamiana epidermal cells coexpressing ERD2-CFP and 6K-YFP. (B) Confocal images of N. benthamiana epidermal cells coexpressing Arf1-CFP and 6K-YFP. (C) High-magnification time-lapse imaging of a cell coexpressing Arf1-CFP and 6K-YFP. Note that Golgi stacks and 6K-YFP punctate structures moved together in the cell at all times. The times of acquisition of frames are indicated in the upper corners. The arrowheads indicate the localization of 6K-YFP punctate structures with the Golgi area. The arrows point to the ringlike structure of 6K-YFP that did not overlap with the Golgi area. Bars, 15 μm.
FIG. 4.
FIG. 4.
Coexpression of dominant-negative Sar1 GTP blocks 6K-CFP accumulation on ERES. (A) Cell coexpressing ERD2-CFP and Sar1-YFP. The arrows indicate colocalization of Golgi bodies with ERES stained with Sar1-YFP. The arrowheads show rare Sar1-YFP punctate labeling that did not overlap with the Golgi stack. (B) Coexpression of Sar1(H74L) led to the retention within the ER of ERD2-CFP. (C) Cell coexpressing 6K-CFP and Sar1-YFP. The arrows indicate colocalization of 6K-CFP punctate structures with ERES stained with Sar1-YFP. The arrowheads show ringlike structures of 6K-CFP that did not colocalize with Sar1-YFP punctate labeling. (D) Expression of Sar1(H74L)-YFP resulted in the retention of 6K-CFP in the ER. Bars, 12 μm.
FIG. 5.
FIG. 5.
Coexpression of dominant-negative Arf1 blocks the accumulation of 6K-CFP at the ERES. (A) Cell coexpressing Sar1-CFP and Arf1-YFP. Sar1-CFP localized in the Golgi area (arrows) but not at the additional small Arf1-YFP structures (arrowheads). (B) Coexpression of Arf1(T31N) led to the retention of Sar1-CFP within the ER. (C) Cell coexpression of Arf1(T31N) causes the retention of 6K-CFP within the ER. Bars, 12 μm.
FIG. 6.
FIG. 6.
TEV infection or expression of 6K-CFP reduces the secretion of secYFP. (A to D) Confocal images at low magnification of N. benthamiana epidermal cells expressing secYFP (A), YFP-HDEL (B), secYFP and 6K (C), and secYFP (D) in the presence of viral infection. (E) Relative YFP fluorescence intensities of N. benthamiana epidermal cells expressing secYFP, YFP-HDEL, secYFP plus 6K, and secYFP in the presence of viral infection. The data are means plus standard errors of three independent experiments. Bars, 150 μm.
FIG. 7.
FIG. 7.
Ectopic expression of dominant-negative mutants of Sar1 and Arf1 reduces TEV infection. (A) Confocal microscopy micrographs of N. benthamiana leaves that were infiltrated with Agrobacterium strains carrying plasmids encoding either Sar1, Sar1(H74L), Arf1, or Arf1(T31N) 24 h before TEV-GFP infection. The photographs show the typical conditions of TEV-GFP replication expressed as the different sizes of GFP foci 3 days postinfection in the absence or presence of the expression of Sar1, Sar1(H74L), Arf1, or Arf1(T31N), respectively. GFP foci are shown in green, and chloroplast autofluorescence is shown in red. Bars, 300 μm. (B) Quantification of the effect of either Sar1, Sar1(H74L), Arf1, or Arf1(T31N) expression upon TEV-GFP replication expressed as the relative average areas of GFP foci with standard errors (SE). The data are means plus SE of three independent experiments. (C) Effect of transient expression of Sar1, Sar1(H74L), Arf1, or Arf1(T31N) on TEV-GFP infection in N. benthamiana. Leaves were infected with TEV 24 h after agroinfiltration and assayed for accumulation of TEV by ELISA. The values represent means plus SE and are given as the percentage of the control.
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