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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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Disruption of the ER induces changes in the localization of wt vRNA. In protoplasts treated at early stages of infection with 50 μg/ml of BFA, the vRNA was accumulated in small fluorescent spots dispersed throughout the cytoplasm (vRNA/BFA). Note the absence of the perinuclear fluorescent structures that accumulate in nontreated protoplasts (see Fig. 2, A–C, for comparisons). N, nucleus. Bar, 10 μm.
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Figure 8: Disruption of the ER induces changes in the localization of wt vRNA. In protoplasts treated at early stages of infection with 50 μg/ml of BFA, the vRNA was accumulated in small fluorescent spots dispersed throughout the cytoplasm (vRNA/BFA). Note the absence of the perinuclear fluorescent structures that accumulate in nontreated protoplasts (see Fig. 2, A–C, for comparisons). N, nucleus. Bar, 10 μm.

Mentions: In protoplasts treated at early stages of infection with 50 μg/ml of Brefeldin A (BFA), a fungal metabolite that disrupts the endomembrane system in plant cells (Henderson et al. 1994; Satiat-Jeunemaitre et al. 1996), the vRNA was dispersed throughout the cytoplasm in small fluorescent spots. No fluorescent vesicle-like structures were observed surrounding the nucleus in treated samples (Fig. 8). These results corroborate the hypothesis that at early stages of infection vRNA is associated with 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)

Disruption of the ER induces changes in the localization of wt vRNA. In protoplasts treated at early stages of infection with 50 μg/ml of BFA, the vRNA was accumulated in small fluorescent spots dispersed throughout the cytoplasm (vRNA/BFA). Note the absence of the perinuclear fluorescent structures that accumulate in nontreated protoplasts (see Fig. 2, A–C, for comparisons). N, nucleus. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169346&req=5

Figure 8: Disruption of the ER induces changes in the localization of wt vRNA. In protoplasts treated at early stages of infection with 50 μg/ml of BFA, the vRNA was accumulated in small fluorescent spots dispersed throughout the cytoplasm (vRNA/BFA). Note the absence of the perinuclear fluorescent structures that accumulate in nontreated protoplasts (see Fig. 2, A–C, for comparisons). N, nucleus. Bar, 10 μm.
Mentions: In protoplasts treated at early stages of infection with 50 μg/ml of Brefeldin A (BFA), a fungal metabolite that disrupts the endomembrane system in plant cells (Henderson et al. 1994; Satiat-Jeunemaitre et al. 1996), the vRNA was dispersed throughout the cytoplasm in small fluorescent spots. No fluorescent vesicle-like structures were observed surrounding the nucleus in treated samples (Fig. 8). These results corroborate the hypothesis that at early stages of infection vRNA is associated with 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