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Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.

Más P, Beachy RN - J. Cell Biol. (1999)

Bottom Line: At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER.Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection.MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

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vRNA colocalizes with the MP. Protoplasts infected with vRNA-MP:GFP-ΔC (see Fig. 1) were fixed at midstages of infection, and the pattern of MP accumulation was observed as fluorescence emitted by the MP:GFP. The samples were then treated to eliminate fluorescence by GFP and hybridized with the fluor-RNA probe to detect vRNA. A cell showing the MP distribution was identified by its position and shape and visualized to detect the products of hybridization (vRNA). Comparison of both images reflects colocalization of MP:GFP and vRNA. N, nucleus. Bars, 10 μm.
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Figure 5: vRNA colocalizes with the MP. Protoplasts infected with vRNA-MP:GFP-ΔC (see Fig. 1) were fixed at midstages of infection, and the pattern of MP accumulation was observed as fluorescence emitted by the MP:GFP. The samples were then treated to eliminate fluorescence by GFP and hybridized with the fluor-RNA probe to detect vRNA. A cell showing the MP distribution was identified by its position and shape and visualized to detect the products of hybridization (vRNA). Comparison of both images reflects colocalization of MP:GFP and vRNA. N, nucleus. Bars, 10 μm.

Mentions: After hybridization with the fluor-RNA probe, the samples were examined to detect the products of hybridization (vRNA). As shown in Fig. 5, there was a striking coincidence between the distribution of MP and the sites of vRNA accumulation. vRNA was also found in small cytoplasmic patches that lacked detectable MP (arrows). Similar patterns of distribution of replicase, MP, and vRNA distribution confirmed the hypothesis that the replication of vRNA and the accumulation of the MP occur at the same subcellular sites.


Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.

Más P, Beachy RN - J. Cell Biol. (1999)

vRNA colocalizes with the MP. Protoplasts infected with vRNA-MP:GFP-ΔC (see Fig. 1) were fixed at midstages of infection, and the pattern of MP accumulation was observed as fluorescence emitted by the MP:GFP. The samples were then treated to eliminate fluorescence by GFP and hybridized with the fluor-RNA probe to detect vRNA. A cell showing the MP distribution was identified by its position and shape and visualized to detect the products of hybridization (vRNA). Comparison of both images reflects colocalization of MP:GFP and vRNA. N, nucleus. Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169346&req=5

Figure 5: vRNA colocalizes with the MP. Protoplasts infected with vRNA-MP:GFP-ΔC (see Fig. 1) were fixed at midstages of infection, and the pattern of MP accumulation was observed as fluorescence emitted by the MP:GFP. The samples were then treated to eliminate fluorescence by GFP and hybridized with the fluor-RNA probe to detect vRNA. A cell showing the MP distribution was identified by its position and shape and visualized to detect the products of hybridization (vRNA). Comparison of both images reflects colocalization of MP:GFP and vRNA. N, nucleus. Bars, 10 μm.
Mentions: After hybridization with the fluor-RNA probe, the samples were examined to detect the products of hybridization (vRNA). As shown in Fig. 5, there was a striking coincidence between the distribution of MP and the sites of vRNA accumulation. vRNA was also found in small cytoplasmic patches that lacked detectable MP (arrows). Similar patterns of distribution of replicase, MP, and vRNA distribution confirmed the hypothesis that the replication of vRNA and the accumulation of the MP occur at the same subcellular sites.

Bottom Line: At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER.Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection.MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

Show MeSH
Related in: MedlinePlus