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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 replicase. Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using antireplicase antibody and in situ hybridization for detecting vRNA. Most of the replicase (red signal) was localized in sites were vRNA (green signal) accumulated. Merging both images demonstrates colocalization of both signals (yellow in merged image). N, nucleus. Bars, 10 μm.
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Figure 4: vRNA colocalizes with the replicase. Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using antireplicase antibody and in situ hybridization for detecting vRNA. Most of the replicase (red signal) was localized in sites were vRNA (green signal) accumulated. Merging both images demonstrates colocalization of both signals (yellow in merged image). N, nucleus. Bars, 10 μm.

Mentions: To examine the relationship between the sites of vRNA accumulation and the location of the viral replicase, protoplasts infected with wt TMV RNA were collected at early stages of infection, first immunostained with antibody raised against the TMV replicase protein (Nelson et al. 1993), and subsequently processed for in situ hybridization with the fluor-RNA probe. The distribution of the replicase, visualized with TRITC-conjugated secondary antibody (Fig. 4, red) was similar to that of vRNA (green). Superimposition of coincident green and red signals are presented in yellow (merged image) and are most apparent in bodies and filaments in the cytoplasm. The antireplicase antibody also labeled structures that lacked vRNA (red in merged image). It is not known whether these differences reflect differences in distribution of replicase protein and vRNA or differences in intensity of each fluorophore.


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 replicase. Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using antireplicase antibody and in situ hybridization for detecting vRNA. Most of the replicase (red signal) was localized in sites were vRNA (green signal) accumulated. Merging both images demonstrates colocalization of both signals (yellow in merged image). N, nucleus. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169346&req=5

Figure 4: vRNA colocalizes with the replicase. Protoplasts infected with wt vRNA were fixed at early stages of infection and processed for immunofluorescence using antireplicase antibody and in situ hybridization for detecting vRNA. Most of the replicase (red signal) was localized in sites were vRNA (green signal) accumulated. Merging both images demonstrates colocalization of both signals (yellow in merged image). N, nucleus. Bars, 10 μm.
Mentions: To examine the relationship between the sites of vRNA accumulation and the location of the viral replicase, protoplasts infected with wt TMV RNA were collected at early stages of infection, first immunostained with antibody raised against the TMV replicase protein (Nelson et al. 1993), and subsequently processed for in situ hybridization with the fluor-RNA probe. The distribution of the replicase, visualized with TRITC-conjugated secondary antibody (Fig. 4, red) was similar to that of vRNA (green). Superimposition of coincident green and red signals are presented in yellow (merged image) and are most apparent in bodies and filaments in the cytoplasm. The antireplicase antibody also labeled structures that lacked vRNA (red in merged image). It is not known whether these differences reflect differences in distribution of replicase protein and vRNA or differences in intensity of each fluorophore.

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