Intracellular localization of the peanut clump virus replication complex in tobacco BY-2 protoplasts containing green fluorescent protein-labeled endoplasmic reticulum or Golgi apparatus

Abstract

RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5'-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.

FIG. 1.

Time course of P131 and P191 synthesis in BY-2 tobacco protoplasts. Protein extracts of mock-infected (lanes 1 and 7) or PCV-infected protoplasts (lanes 2 to 5 and lanes 8 to 12) harvested at the time (hours) postinfection indicated at the bottom were analyzed by Western blotting. The proteins were separated by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis, transferred onto an Immobilon-P membrane, and probed with antisera raised against P131 (lanes 1 to 5) or P191 (lanes 7 to 10) or with affinity chromatography-purified anti-P131 (lane 11) or anti-P191 (lane 12) antibodies. Bound antibodies were visualized by chemiluminescence. Lane 6 correponds to in vitro [³⁵S]methionine-labeled translation products obtained after incubation of viral PCV RNA in a wheat germ extract. The positions of P131 and P191 are indicated by arrows.

FIG. 2.

(Top). Localization of Br-RNA and dsRNA in typical PCV-infected BY-2 tobacco protoplasts. PCV-infected (A, B, and C) or mock-inoculated (D and E) protoplasts were treated with actinomycin D added at 17 hpi and then incubated for 1 h prior to addition of BrUTP and further incubated for 6 h. The protoplasts were then immunolabeled with mouse BrUTP-specific antibodies and dsRNA guinea pig antibodies, which were then detected with, respectively, Alexa 488 (A and D)- and Alexa 568 (B and E)-labeled secondary antibodies. BrUTP labeling is in green, and dsRNA labeling is in red. (C) Superimposition of the images to the left. The confocal images were collected with a focal depth of 0.45 μm. Bar, 10 μm.

FIG. 3.

(Bottom). Colocalization of incorporated BrUTP or dsRNA with P131 or P191 in BY-2 protoplasts. PCV-infected protoplasts were processed for double-label immunofluorescence. P131 (A and D) and P191 (G and J) were detected by indirect immunofluorescence (in green) with purified rabbit antibodies raised against P131 or P191. The distribution of BrUTP (B and H) at 24 hpi and of dsRNA (E and K) at 48 h pi was imaged in the same 0.45-μm optical section as for P131 and P191 and is shown in red. The right column (C, F, I, and L) shows digital superimposition of the two panels to the left. Bar, 10 μm.

FIG. 4.

(Left). Localization of P131 and P191 in transgenic BY-2 protoplasts. Healthy (A and B) or PCV-infected (C to H) ER-GFP protoplasts expressing a GFP targeted to the ER were processed for fluorescence with primary antiserum against P131 at 24 hpi (B and D) or against P191 at 48 hpi (G). Similarly, healthy (I and J) or infected (K to P) Man1::GFP protoplasts expressing a GFP targeted to the Golgi apparatus were processed to visualize the P131 (J and L) or the P191 (O) accumulation sites. The digital superimposition of both fluorescent signals is shown in panels E, H, M, and P. Bar, 10 μm.

FIG. 5.

(Right). Localization of P15 compared with that of Br-RNA, P131, and ER. The first row of images shows the same ER-GFP transgenic protoplast infected with T1, T2, and TRep-EG15 processed at 24 hpi. Green fluorescence of EGFP/P15 fusion protein is shown in panel A. Primary antibodies against Br-RNA and P131 were revealed, respectively, by Alexa 568 (red, B) and Alexa 633 (white, C) secondary antibody. Digital superimposition of images from the same 0.45-μm optical section shows Br-RNA and EGFP/P15 (D), P131 and EGFP/P15 (E), and P131 and Br-RNA (F). The third and fourth rows correspond to the same representative ER-GFP transgenic protoplast infected with T1, T2, and TRep-RFP15 (red) in two different optical sections at 48 hpi. Green ER-GFP fluorescence (G and J) and red RFP/P15 fluorescence (H and K) are superimposed in panels I and L. Bar, 10 μm.

FIG. 6.

Analysis of the accumulation of viral RNA in drug-treated PCV-infected protoplasts. BY-2 tobacco protoplasts inoculated with PCV RNA were either untreated (lanes 1 and 5) or treated with 15, 30, or 45 μM cerulenin (lanes 2, 3, and 4) or with 10 or 30 μg of BFA/ml (lanes 6 and 7). The drug was added just after infection, and the protoplasts were harvested at 48 hpi. H corresponds to total RNA extracted from uninfected protoplasts. Blots of extracted total RNA were probed with a ³²P-labeled riboprobe corresponding to the complementary sequence of 3′-terminal 124 nucleotides common to both PCV genomic RNAs. The positions of RNA-1 and RNA-2 are indicated by arrows.

FIG. 7.

Confocal fluorescence micrograph of mock-infected (A and B) or PCV-infected (C and D) epidermal cells of ER-GFP transgenic N. benthamiana. (A) Perinuclear and plasma membranes show a fluorescent halo in a healthy ER-GFP N. benthamiana. (B) Normal reticulate pattern of cortical ER in apical leaf cell of mock-inoculated plants. (C) Perinuclear ER aggregates (arrow) observed in epidermal cells of PCV-infected systemic leaves. (D) Cortical fluorescent bodies and typical ER network in infected leaves. Bar, 10 μm.

FIG. 8.

Electron microscopy of cytopathological structures in PCV-infected BY-2 protoplasts at 48 hpi. (A) Low-magnification view of the ultrastructural modifications. (B, C, D, E, and F) Higher-magnification views of the different cytopathic structures. Black asterisks correspond to area of broken ER, black arrows point to constrictions on the ER fragments, and white arrows indicate clusters of vesicles. Single arrowheads correspond to MVB; MVB containing disordered membranous vesicles are indicated by black arrowheads, whereas those containing one row of vesicles and surrounded by a single membrane are indicated by white arrowheads. White asterisks correspond to electron-dense material without detectable vesicles. Double arrows indicate clusters of virions. (F) Magnification of the inset square indicated in panel A. N, nucleus. Bars, 1 μm in panel A and 0.5 μm in panels B, C, D, E, and F.