Limits...
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.

Show MeSH

Related in: MedlinePlus

Disruption of cytoskeleton induces changes in the localization of wt vRNA and vRNA-ΔM. Protoplasts treated at midstages of infection with 10 μM oryzalin for 2 h induces the accumulation of vRNA in enlarged fluorescent bodies surrounding the nucleus (vRNA/Oryzalin). Treatment at early stages of infection with cytochalasin D delays the formation of large bodies, and the pattern of vRNA accumulation was similar to that observed in infection by vRNA-ΔM (vRNA/Cytoch D). In protoplasts infected with vRNA-ΔM and treated at early stages of infection with cytochalasin D, vRNA-ΔM accumulated in filaments and narrow structures at the periphery of the cell (vRNA-ΔM/Cytoch D). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169346&req=5

Figure 10: Disruption of cytoskeleton induces changes in the localization of wt vRNA and vRNA-ΔM. Protoplasts treated at midstages of infection with 10 μM oryzalin for 2 h induces the accumulation of vRNA in enlarged fluorescent bodies surrounding the nucleus (vRNA/Oryzalin). Treatment at early stages of infection with cytochalasin D delays the formation of large bodies, and the pattern of vRNA accumulation was similar to that observed in infection by vRNA-ΔM (vRNA/Cytoch D). In protoplasts infected with vRNA-ΔM and treated at early stages of infection with cytochalasin D, vRNA-ΔM accumulated in filaments and narrow structures at the periphery of the cell (vRNA-ΔM/Cytoch D). Bars, 10 μm.

Mentions: The results provide strong evidence for colocalization of vRNA with microtubules and suggest that cytoskeletal elements may be involved in distribution of vRNA in protoplasts. To clarify the role of the cytoskeleton in vRNA distribution, protoplasts infected with wt vRNA were treated with specific cytoskeletal inhibitors and vRNA accumulation was examined by in situ hybridization. Representative examples are shown in Fig. 10. Treatment of protoplasts at early stages of infection with oryzalin, a plant microtubule depolymerizing agent (Hugdahl and Morejohn 1993), abolished the accumulation of vRNA around the nucleus and most vRNA was dispersed throughout the cytoplasm (not shown). When oryzalin was added at midstages of infection, nearly all of the fluorescence was associated with enlarged fluorescent bodies on or near the nuclear envelope (Fig. 10, vRNA/Oryzalin). In nontreated protoplasts, most of the fluorescent bodies were dispersed throughout the cytoplasm at this stage of infection (e.g., Fig. 2 G). When oryzalin was added late in infection, we did not observe changes in distribution of vRNA. In treated as well as nontreated protoplasts, vRNA was associated with the filament-like structures protruding from the surface of the cells (see Fig. 2 K).


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 cytoskeleton induces changes in the localization of wt vRNA and vRNA-ΔM. Protoplasts treated at midstages of infection with 10 μM oryzalin for 2 h induces the accumulation of vRNA in enlarged fluorescent bodies surrounding the nucleus (vRNA/Oryzalin). Treatment at early stages of infection with cytochalasin D delays the formation of large bodies, and the pattern of vRNA accumulation was similar to that observed in infection by vRNA-ΔM (vRNA/Cytoch D). In protoplasts infected with vRNA-ΔM and treated at early stages of infection with cytochalasin D, vRNA-ΔM accumulated in filaments and narrow structures at the periphery of the cell (vRNA-ΔM/Cytoch D). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Disruption of cytoskeleton induces changes in the localization of wt vRNA and vRNA-ΔM. Protoplasts treated at midstages of infection with 10 μM oryzalin for 2 h induces the accumulation of vRNA in enlarged fluorescent bodies surrounding the nucleus (vRNA/Oryzalin). Treatment at early stages of infection with cytochalasin D delays the formation of large bodies, and the pattern of vRNA accumulation was similar to that observed in infection by vRNA-ΔM (vRNA/Cytoch D). In protoplasts infected with vRNA-ΔM and treated at early stages of infection with cytochalasin D, vRNA-ΔM accumulated in filaments and narrow structures at the periphery of the cell (vRNA-ΔM/Cytoch D). Bars, 10 μm.
Mentions: The results provide strong evidence for colocalization of vRNA with microtubules and suggest that cytoskeletal elements may be involved in distribution of vRNA in protoplasts. To clarify the role of the cytoskeleton in vRNA distribution, protoplasts infected with wt vRNA were treated with specific cytoskeletal inhibitors and vRNA accumulation was examined by in situ hybridization. Representative examples are shown in Fig. 10. Treatment of protoplasts at early stages of infection with oryzalin, a plant microtubule depolymerizing agent (Hugdahl and Morejohn 1993), abolished the accumulation of vRNA around the nucleus and most vRNA was dispersed throughout the cytoplasm (not shown). When oryzalin was added at midstages of infection, nearly all of the fluorescence was associated with enlarged fluorescent bodies on or near the nuclear envelope (Fig. 10, vRNA/Oryzalin). In nontreated protoplasts, most of the fluorescent bodies were dispersed throughout the cytoplasm at this stage of infection (e.g., Fig. 2 G). When oryzalin was added late in infection, we did not observe changes in distribution of vRNA. In treated as well as nontreated protoplasts, vRNA was associated with the filament-like structures protruding from the surface of the cells (see Fig. 2 K).

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