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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 BiP (ER marker). Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using anti–BiP antibody and in situ hybridization. Most of the BiP (red) was associated with sites that contain vRNA (green). Merging the two images demonstrates colocalization of the signals (yellow in merged image). Bars, 10 μm.
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Figure 7: vRNA colocalizes with BiP (ER marker). Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using anti–BiP antibody and in situ hybridization. Most of the BiP (red) was associated with sites that contain vRNA (green). Merging the two images demonstrates colocalization of the signals (yellow in merged image). Bars, 10 μm.

Mentions: As noted above, the reticulated pattern of fluorescence that represents the location of wt vRNA resembled the distribution of the ER. Furthermore, previous studies demonstrated colocalization of TMV MP and the replicase with ER on the fluorescent irregularly shaped bodies (Heinlein et al. 1998; Reichel and Beachy 1998). To determine if vRNA is associated with the ER, in situ hybridization was performed on protoplasts collected at early stages of infection. The samples were also stained using an antibody to the ER BiP (Boston et al. 1996) and a TRITC-conjugated secondary antibody. As shown in Fig. 7, BiP was localized to perinuclear areas and small aggregates dispersed in the cytoplasm. vRNA was localized in structures surrounding the nucleus and scattered in the cytoplasm and in fluorescent bodies. After superimposing the images, we concluded that a portion of the vRNA detected is associated with the ER (yellow in merged image). BiP and vRNA were not fully coincident, indicating that a fraction of vRNA was independent of the ER.


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 BiP (ER marker). Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using anti–BiP antibody and in situ hybridization. Most of the BiP (red) was associated with sites that contain vRNA (green). Merging the two images demonstrates colocalization of the signals (yellow in merged image). Bars, 10 μm.
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Related In: Results  -  Collection

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

Figure 7: vRNA colocalizes with BiP (ER marker). Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using anti–BiP antibody and in situ hybridization. Most of the BiP (red) was associated with sites that contain vRNA (green). Merging the two images demonstrates colocalization of the signals (yellow in merged image). Bars, 10 μm.
Mentions: As noted above, the reticulated pattern of fluorescence that represents the location of wt vRNA resembled the distribution of the ER. Furthermore, previous studies demonstrated colocalization of TMV MP and the replicase with ER on the fluorescent irregularly shaped bodies (Heinlein et al. 1998; Reichel and Beachy 1998). To determine if vRNA is associated with the ER, in situ hybridization was performed on protoplasts collected at early stages of infection. The samples were also stained using an antibody to the ER BiP (Boston et al. 1996) and a TRITC-conjugated secondary antibody. As shown in Fig. 7, BiP was localized to perinuclear areas and small aggregates dispersed in the cytoplasm. vRNA was localized in structures surrounding the nucleus and scattered in the cytoplasm and in fluorescent bodies. After superimposing the images, we concluded that a portion of the vRNA detected is associated with the ER (yellow in merged image). BiP and vRNA were not fully coincident, indicating that a fraction of vRNA was independent of the ER.

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