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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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Intracellular distribution of vRNA in protoplasts infected with wt vRNA. Protoplasts were collected at different times after infection, fixed, and hybridized with a fluorescein-RNA probe that recognized TMV RNA. Fluorescent signals were visualized by confocal microscopy, collecting optical sections with a focal depth of 0.8 μm. (A–C) At early stages of infection (6–12 hpi), vRNA is associated with vesicle-like structures surrounding the nucleus (A) and in small patches or particles in the cytoplasm (B and C). Note the large amount of vRNA in perinuclear regions (C). (D–I) vRNA at midstages of infection (12–22 hpi) localized in large and small bodies (D and E), a weakly fluorescent reticulated network (E), large vesicles (F), large bodies (G) that appear to be associated with filaments (H and I). (J and K) vRNA at late stages of infection (22–36 hpi). vRNA is associated with small, intensely labeled spots around the nucleus, dispersed throughout the cytoplasm, and at the periphery of the cell (J) and localized in filamentous structures, some of which protrude from the surface of the cells (K). (L) Mock-inoculated protoplasts identically processed and imaged. The images shown in A, B, K, and L correspond to projections of serial optical sections, while C, D, F–H, and J represent single optical sections. Bars: (A–D and F–G) 10 μm; (E–I) 5 μm.
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Figure 2: Intracellular distribution of vRNA in protoplasts infected with wt vRNA. Protoplasts were collected at different times after infection, fixed, and hybridized with a fluorescein-RNA probe that recognized TMV RNA. Fluorescent signals were visualized by confocal microscopy, collecting optical sections with a focal depth of 0.8 μm. (A–C) At early stages of infection (6–12 hpi), vRNA is associated with vesicle-like structures surrounding the nucleus (A) and in small patches or particles in the cytoplasm (B and C). Note the large amount of vRNA in perinuclear regions (C). (D–I) vRNA at midstages of infection (12–22 hpi) localized in large and small bodies (D and E), a weakly fluorescent reticulated network (E), large vesicles (F), large bodies (G) that appear to be associated with filaments (H and I). (J and K) vRNA at late stages of infection (22–36 hpi). vRNA is associated with small, intensely labeled spots around the nucleus, dispersed throughout the cytoplasm, and at the periphery of the cell (J) and localized in filamentous structures, some of which protrude from the surface of the cells (K). (L) Mock-inoculated protoplasts identically processed and imaged. The images shown in A, B, K, and L correspond to projections of serial optical sections, while C, D, F–H, and J represent single optical sections. Bars: (A–D and F–G) 10 μm; (E–I) 5 μm.

Mentions: The intracellular accumulation of TMV RNA during infection was visualized by in situ hybridization. Protoplasts isolated from BY-2 tobacco cells were transfected with wt RNA, collected at different times after infection, and hybridized with fluor-RNA probes. The fluorescent signals were visualized by confocal microscopy after collecting optical sections with a focal depth of 0.8 μm. Since infection of BY-2 protoplasts is apparently not synchronous (Heinlein et al. 1998), the accumulation of vRNA was considered in three stages: early, 6–12 hpi; mid, 12–22 hpi; and late, 22–36 hpi. Representative examples are shown in Fig. 2. At early stages of infection (Fig. 2, A–C), vRNA was primarily localized in faint fluorescent vesicle-like structures surrounding the nucleus (Fig. 2 A). The fluorescent signal was also observed in small, discrete cytoplasmic patches of uniform size, dispersed randomly throughout the cell (Fig. 2 B, arrowheads). In some cells, vRNA tended to accumulate in large amounts around the nucleus as shown in an optical section in Fig. 2 C.


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 vRNA in protoplasts infected with wt vRNA. Protoplasts were collected at different times after infection, fixed, and hybridized with a fluorescein-RNA probe that recognized TMV RNA. Fluorescent signals were visualized by confocal microscopy, collecting optical sections with a focal depth of 0.8 μm. (A–C) At early stages of infection (6–12 hpi), vRNA is associated with vesicle-like structures surrounding the nucleus (A) and in small patches or particles in the cytoplasm (B and C). Note the large amount of vRNA in perinuclear regions (C). (D–I) vRNA at midstages of infection (12–22 hpi) localized in large and small bodies (D and E), a weakly fluorescent reticulated network (E), large vesicles (F), large bodies (G) that appear to be associated with filaments (H and I). (J and K) vRNA at late stages of infection (22–36 hpi). vRNA is associated with small, intensely labeled spots around the nucleus, dispersed throughout the cytoplasm, and at the periphery of the cell (J) and localized in filamentous structures, some of which protrude from the surface of the cells (K). (L) Mock-inoculated protoplasts identically processed and imaged. The images shown in A, B, K, and L correspond to projections of serial optical sections, while C, D, F–H, and J represent single optical sections. Bars: (A–D and F–G) 10 μm; (E–I) 5 μm.
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Figure 2: Intracellular distribution of vRNA in protoplasts infected with wt vRNA. Protoplasts were collected at different times after infection, fixed, and hybridized with a fluorescein-RNA probe that recognized TMV RNA. Fluorescent signals were visualized by confocal microscopy, collecting optical sections with a focal depth of 0.8 μm. (A–C) At early stages of infection (6–12 hpi), vRNA is associated with vesicle-like structures surrounding the nucleus (A) and in small patches or particles in the cytoplasm (B and C). Note the large amount of vRNA in perinuclear regions (C). (D–I) vRNA at midstages of infection (12–22 hpi) localized in large and small bodies (D and E), a weakly fluorescent reticulated network (E), large vesicles (F), large bodies (G) that appear to be associated with filaments (H and I). (J and K) vRNA at late stages of infection (22–36 hpi). vRNA is associated with small, intensely labeled spots around the nucleus, dispersed throughout the cytoplasm, and at the periphery of the cell (J) and localized in filamentous structures, some of which protrude from the surface of the cells (K). (L) Mock-inoculated protoplasts identically processed and imaged. The images shown in A, B, K, and L correspond to projections of serial optical sections, while C, D, F–H, and J represent single optical sections. Bars: (A–D and F–G) 10 μm; (E–I) 5 μm.
Mentions: The intracellular accumulation of TMV RNA during infection was visualized by in situ hybridization. Protoplasts isolated from BY-2 tobacco cells were transfected with wt RNA, collected at different times after infection, and hybridized with fluor-RNA probes. The fluorescent signals were visualized by confocal microscopy after collecting optical sections with a focal depth of 0.8 μm. Since infection of BY-2 protoplasts is apparently not synchronous (Heinlein et al. 1998), the accumulation of vRNA was considered in three stages: early, 6–12 hpi; mid, 12–22 hpi; and late, 22–36 hpi. Representative examples are shown in Fig. 2. At early stages of infection (Fig. 2, A–C), vRNA was primarily localized in faint fluorescent vesicle-like structures surrounding the nucleus (Fig. 2 A). The fluorescent signal was also observed in small, discrete cytoplasmic patches of uniform size, dispersed randomly throughout the cell (Fig. 2 B, arrowheads). In some cells, vRNA tended to accumulate in large amounts around the nucleus as shown in an optical section in Fig. 2 C.

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