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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 microtubules. Protoplasts infected with wt vRNA were fixed and processed for immunofluorescence using antitubulin antibody and in situ hybridization. (A) Most of the tubulin (red) was associated with filaments that coaligned with the filaments that contain vRNA (green). Merging the images demonstrates colocalization of the signals (yellow in merged image). (B) Some of the bright fluorescent patches containing vRNA (green) appear to be aligned with microtubule filaments (red). Bars, (A) 10 μm; (B) 2 μm.
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Figure 9: vRNA colocalizes with microtubules. Protoplasts infected with wt vRNA were fixed and processed for immunofluorescence using antitubulin antibody and in situ hybridization. (A) Most of the tubulin (red) was associated with filaments that coaligned with the filaments that contain vRNA (green). Merging the images demonstrates colocalization of the signals (yellow in merged image). (B) Some of the bright fluorescent patches containing vRNA (green) appear to be aligned with microtubule filaments (red). Bars, (A) 10 μm; (B) 2 μm.

Mentions: At early and midstages of infection, vRNA was associated with fluorescent cytoplasmic filaments (Fig. 2H and Fig. I). To determine if vRNA is associated with elements of the cytoskeleton, infected protoplasts that were immunostained with a monoclonal antibody to α-tubulin were processed for in situ hybridization. A variety of different experimental conditions was evaluated before selecting conditions that permitted visualization of both signals. The procedure that was used involved treating fixed protoplasts with 1 μg/ml of proteinase K for 5 min to partially digest cross-linked proteins and subsequent refixation to avoid disintegration of the cells. These procedures improved the accessibility of the probe while preserving the integrity of the microtubules. Fig. 9 shows confocal micrographs of tubulin (in red) and vRNA (in green) in an infected protoplast processed at midstage of infection. Merging the images clearly showed the coalignment of both signals (merged image). Most of the green fluorescent filaments corresponding to sites of vRNA accumulation coaligned with the cytoplasmic strands of microtubules (Fig. 9 A, yellow in merged image). Furthermore, some of the bright fluorescent spots that contained vRNA accumulated in tracks that were aligned with microtubule filaments (Fig. 9 B).


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 microtubules. Protoplasts infected with wt vRNA were fixed and processed for immunofluorescence using antitubulin antibody and in situ hybridization. (A) Most of the tubulin (red) was associated with filaments that coaligned with the filaments that contain vRNA (green). Merging the images demonstrates colocalization of the signals (yellow in merged image). (B) Some of the bright fluorescent patches containing vRNA (green) appear to be aligned with microtubule filaments (red). Bars, (A) 10 μm; (B) 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169346&req=5

Figure 9: vRNA colocalizes with microtubules. Protoplasts infected with wt vRNA were fixed and processed for immunofluorescence using antitubulin antibody and in situ hybridization. (A) Most of the tubulin (red) was associated with filaments that coaligned with the filaments that contain vRNA (green). Merging the images demonstrates colocalization of the signals (yellow in merged image). (B) Some of the bright fluorescent patches containing vRNA (green) appear to be aligned with microtubule filaments (red). Bars, (A) 10 μm; (B) 2 μm.
Mentions: At early and midstages of infection, vRNA was associated with fluorescent cytoplasmic filaments (Fig. 2H and Fig. I). To determine if vRNA is associated with elements of the cytoskeleton, infected protoplasts that were immunostained with a monoclonal antibody to α-tubulin were processed for in situ hybridization. A variety of different experimental conditions was evaluated before selecting conditions that permitted visualization of both signals. The procedure that was used involved treating fixed protoplasts with 1 μg/ml of proteinase K for 5 min to partially digest cross-linked proteins and subsequent refixation to avoid disintegration of the cells. These procedures improved the accessibility of the probe while preserving the integrity of the microtubules. Fig. 9 shows confocal micrographs of tubulin (in red) and vRNA (in green) in an infected protoplast processed at midstage of infection. Merging the images clearly showed the coalignment of both signals (merged image). Most of the green fluorescent filaments corresponding to sites of vRNA accumulation coaligned with the cytoplasmic strands of microtubules (Fig. 9 A, yellow in merged image). Furthermore, some of the bright fluorescent spots that contained vRNA accumulated in tracks that were aligned with microtubule filaments (Fig. 9 B).

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