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

Intracellular distribution of TMV minus-strand RNA in protoplasts infected with wt RNA. Protoplasts collected at early stages of infection (4–6 hpi) were fixed and hybridized with a fluorescein-RNA probe that recognized the complementary strand of vRNA. (A) Minus-strand RNA accumulation in a perinuclear pattern and in small cytoplasmic patches. (B) RNA surrounding the nucleus with a discrete region that lacked hybridization. (C and D) Nuclei of protoplasts at 6 hpi showing vRNA-free zone. A, C, and D correspond to single optical sections, while B corresponds to a projection of serial optical sections. N, nucleus. Bars: (A and B) 10 μm; (C and D) 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169346&req=5

Figure 3: Intracellular distribution of TMV minus-strand RNA in protoplasts infected with wt RNA. Protoplasts collected at early stages of infection (4–6 hpi) were fixed and hybridized with a fluorescein-RNA probe that recognized the complementary strand of vRNA. (A) Minus-strand RNA accumulation in a perinuclear pattern and in small cytoplasmic patches. (B) RNA surrounding the nucleus with a discrete region that lacked hybridization. (C and D) Nuclei of protoplasts at 6 hpi showing vRNA-free zone. A, C, and D correspond to single optical sections, while B corresponds to a projection of serial optical sections. N, nucleus. Bars: (A and B) 10 μm; (C and D) 5 μm.

Mentions: As anticipated from the Northern hybridization studies described above and those described by Ishikawa et al. 1991, in situ hybridization experiments using a probe that bound minus-strand RNA revealed fluorescent signals only at early stages of infection (4–8 hpi). As illustrated in Fig. 3, fluorescence accumulated primarily in perinuclear vesicles and in small cytoplasmic patches, similar to the accumulation of plus-strand vRNA (compare with Fig. 2 B). Interestingly, the surface of the nucleus was not uniformly covered by fluorescence in these reactions. In protoplasts visualized at 4–6 hpi, the fluorescent signal was clearly absent from a discrete region of most nuclei (Fig. 3 B). Visualization at high magnification revealed circular regions free of RNA in protoplasts with weak (Fig. 3 C) or strong (Fig. 3 D) fluorescence around the nucleus.


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)

Intracellular distribution of TMV minus-strand RNA in protoplasts infected with wt RNA. Protoplasts collected at early stages of infection (4–6 hpi) were fixed and hybridized with a fluorescein-RNA probe that recognized the complementary strand of vRNA. (A) Minus-strand RNA accumulation in a perinuclear pattern and in small cytoplasmic patches. (B) RNA surrounding the nucleus with a discrete region that lacked hybridization. (C and D) Nuclei of protoplasts at 6 hpi showing vRNA-free zone. A, C, and D correspond to single optical sections, while B corresponds to a projection of serial optical sections. N, nucleus. Bars: (A and B) 10 μm; (C and D) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Intracellular distribution of TMV minus-strand RNA in protoplasts infected with wt RNA. Protoplasts collected at early stages of infection (4–6 hpi) were fixed and hybridized with a fluorescein-RNA probe that recognized the complementary strand of vRNA. (A) Minus-strand RNA accumulation in a perinuclear pattern and in small cytoplasmic patches. (B) RNA surrounding the nucleus with a discrete region that lacked hybridization. (C and D) Nuclei of protoplasts at 6 hpi showing vRNA-free zone. A, C, and D correspond to single optical sections, while B corresponds to a projection of serial optical sections. N, nucleus. Bars: (A and B) 10 μm; (C and D) 5 μm.
Mentions: As anticipated from the Northern hybridization studies described above and those described by Ishikawa et al. 1991, in situ hybridization experiments using a probe that bound minus-strand RNA revealed fluorescent signals only at early stages of infection (4–8 hpi). As illustrated in Fig. 3, fluorescence accumulated primarily in perinuclear vesicles and in small cytoplasmic patches, similar to the accumulation of plus-strand vRNA (compare with Fig. 2 B). Interestingly, the surface of the nucleus was not uniformly covered by fluorescence in these reactions. In protoplasts visualized at 4–6 hpi, the fluorescent signal was clearly absent from a discrete region of most nuclei (Fig. 3 B). Visualization at high magnification revealed circular regions free of RNA in protoplasts with weak (Fig. 3 C) or strong (Fig. 3 D) fluorescence around the nucleus.

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