Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein - PubMed (original) (raw)

Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein

P Mas et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Despite its central role in virus infection, little is known about the mechanisms of intracellular trafficking of virus components within infected cells. In this study, we followed the dynamics of tobacco mosaic virus movement protein (MP) distribution in living protoplasts after disruption of microtubules (MTs) by cold treatment and subsequent rewarming to 29 degrees C. At early stages of infection, cold treatment (4 degrees C) caused the accumulation of MP fused to green fluorescent protein (GFP) in large virus replication bodies that localized in perinuclear positions, whereas at midstages of infection, the association of MP:GFP with MTs was disrupted. Rewarming the protoplasts to 29 degrees C reestablished the association of MTs with the replication bodies that subsequently spread throughout the cytoplasm and to the periphery of the cell. The role of MTs in the intracellular distribution of the MP also was analyzed by examining the distribution pattern of a nonfunctional mutant of MP (TAD5). Like MP:GFP, TAD5:GFP interacted with the endoplasmic reticulum membranes and colocalized with its viral RNA but did not colocalize with MTs. The involvement of MTs in the intracellular distribution of tobacco mosaic virus MP is discussed.

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Figures

Figure 1

Figure 1

Effects of cold treatment on MT organization in BY-2 protoplasts. (A) Noninoculated protoplasts processed for immunofluorescence by using antitubulin antibody and a secondary antibody labeled with TRITC. (Left) Cytoplasmic filaments of tubulin in cells incubated at 29°C. (Right) Disruption of MT organization in cells maintained at 4°C for 3 h. (Scale bar: 2.5 μm.) (B) Protoplasts infected with TMV MP:GFP. At 18 hpi, MP:GFP is accumulated in replication bodies associated with MTs. (Left) A single protoplast maintained at 29°C for 18 h. (Right) Same protoplast after treatment at 4°C for 2 h. Note the absence of fluorescent filaments after cold treatment. (Scale bar: 5 μm.)

Figure 2

Figure 2

Role of MTs in MP:GFP distribution. Accumulation of MP:GFP in a single-infected protoplast examined after cold treatment at 4°C for 6 h (A) and subsequent rewarming to 29°C for 1 h (B) and 3 h (C). Note that cold treatment induced the localization of replication bodies around the nucleus (A). After rewarming the protoplasts, MP:GFP is closely associated with MTs (B and C). Most of the fluorescent bodies were localized at the periphery of the cell (C). (Scale bar: 2.5 μm.)

Figure 3

Figure 3

TAD5:GFP remains associated with perinuclear replication bodies throughout infection. Accumulation of TAD5:GFP in a single protoplast at 12, 18, 24, and 30 hpi. The images were obtained after overlaying multiple optical confocal images. TAD5:GFP accumulated in replication bodies that localized around the nucleus throughout infection. (Scale bar: 2.5 μm.)

Figure 4

Figure 4

TAD5:GFP colocalizes with BiP (ER marker). BY-2 protoplasts infected with TAD5:GFP were fixed and processed for immunofluorescence by using anti-BiP antibody followed by a secondary antibody labeled with TRITC. Most of the BiP (red) was associated with sites that contain TAD5:GFP (green). Merging the two images demonstrates colocalization of the signals (yellow in merged image). (Scale bar: 2.5 μm.)

Figure 5

Figure 5

TAD5:GFP does not colocalize with MTs. Protoplasts infected with TAD5:GFP were fixed and processed for immunofluorescence by using antitubulin antibody followed by a secondary antibody labeled with TRITC. The cytoplasmic filaments of tubulin (red) did not colocalize with TAD5:GFP (green). Merging the images demonstrates absence of colocalization between the signals (merged image). (Scale bar: 2.5 μm.)

Figure 6

Figure 6

(A) Accumulation of genome-length TMV plus-strand RNA in tobacco protoplasts inoculated with wild-type TMV MP:GFP or TAD5:GFP. Total RNA was extracted at 8, 16, and 24 hpi, glyoxylated, and subjected by electrophoresis on a 1% denaturing agarose gel. Northern blots were probed with a digoxigenin-labeled RNA probe that recognized the plus-strand of TMV RNA. (B) vRNA colocalizes with TAD5:GFP. Protoplasts infected with TAD5:GFP virus were fixed at midstages of infection, and the pattern of TAD5:GFP accumulation was observed by confocal microscopy. The samples were treated to eliminate fluorescence by GFP and hybridized with the fluorescein-RNA probe to detect TAD5 vRNA. A cell showing the TAD5:GFP distribution was identified by its position and shape and visualized to detect the products of hybridization (TAD5 vRNA). Comparison of both images reflects colocalization of TAD5:GFP and vRNA. (Scale bar: 2.5 μm.)

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