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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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(A) Diagrammatic representation of the genomic structures of wt TMV RNA and derivatives used in this study (indicated in the left margin). Coding regions for the replicase, MP, GFP, and CP are shown by open boxes. Broken lines represent fusion proteins (MP fused to GFP), while solid lines represent expression of free proteins. (B) Accumulation of genome-length TMV plus-strand RNA in tobacco protoplasts inoculated with wt TMV RNA (w.t.) and with different TMV derivatives (indicated above each lane). “Mock” corresponds to protoplasts treated in absence of inoculum. Total RNA was extracted at 4, 14, and 30 hpi, glyoxylated, and subjected by electrophoresis on a 1% denaturing agarose gel. Northern blots were probed with a digoxigenin-labeled RNA probe that recognized the plus strand of TMV RNA. Each construct was tested in at least three independent experiments; results consistent with those shown were obtained in each experiment.
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Figure 1: (A) Diagrammatic representation of the genomic structures of wt TMV RNA and derivatives used in this study (indicated in the left margin). Coding regions for the replicase, MP, GFP, and CP are shown by open boxes. Broken lines represent fusion proteins (MP fused to GFP), while solid lines represent expression of free proteins. (B) Accumulation of genome-length TMV plus-strand RNA in tobacco protoplasts inoculated with wt TMV RNA (w.t.) and with different TMV derivatives (indicated above each lane). “Mock” corresponds to protoplasts treated in absence of inoculum. Total RNA was extracted at 4, 14, and 30 hpi, glyoxylated, and subjected by electrophoresis on a 1% denaturing agarose gel. Northern blots were probed with a digoxigenin-labeled RNA probe that recognized the plus strand of TMV RNA. Each construct was tested in at least three independent experiments; results consistent with those shown were obtained in each experiment.

Mentions: Diagrams of the infectious clones used in this study are shown in Fig. 1 A (below). The plasmids pU3/12 (Holt and Beachy 1991) and pU3/12 ΔM-RV (Nejidat et al. 1991) were used to generate infectious transcripts of wild-type (wt) TMV and TMV that does not express a functional MP (ΔM), respectively. The plasmids pT-MfCP (MP:GFP-CP) and pTMV-M:GfusBr (MP:GFP-ΔC) (Oparka et al. 1997) contain the MP fused to GFP in which serine at amino acid position 65 is replaced by threonine (Heim et al. 1995). vRNA-MP:GFP-ΔC does not contain the CP open reading frame (see Fig. 1 A).


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)

(A) Diagrammatic representation of the genomic structures of wt TMV RNA and derivatives used in this study (indicated in the left margin). Coding regions for the replicase, MP, GFP, and CP are shown by open boxes. Broken lines represent fusion proteins (MP fused to GFP), while solid lines represent expression of free proteins. (B) Accumulation of genome-length TMV plus-strand RNA in tobacco protoplasts inoculated with wt TMV RNA (w.t.) and with different TMV derivatives (indicated above each lane). “Mock” corresponds to protoplasts treated in absence of inoculum. Total RNA was extracted at 4, 14, and 30 hpi, glyoxylated, and subjected by electrophoresis on a 1% denaturing agarose gel. Northern blots were probed with a digoxigenin-labeled RNA probe that recognized the plus strand of TMV RNA. Each construct was tested in at least three independent experiments; results consistent with those shown were obtained in each experiment.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) Diagrammatic representation of the genomic structures of wt TMV RNA and derivatives used in this study (indicated in the left margin). Coding regions for the replicase, MP, GFP, and CP are shown by open boxes. Broken lines represent fusion proteins (MP fused to GFP), while solid lines represent expression of free proteins. (B) Accumulation of genome-length TMV plus-strand RNA in tobacco protoplasts inoculated with wt TMV RNA (w.t.) and with different TMV derivatives (indicated above each lane). “Mock” corresponds to protoplasts treated in absence of inoculum. Total RNA was extracted at 4, 14, and 30 hpi, glyoxylated, and subjected by electrophoresis on a 1% denaturing agarose gel. Northern blots were probed with a digoxigenin-labeled RNA probe that recognized the plus strand of TMV RNA. Each construct was tested in at least three independent experiments; results consistent with those shown were obtained in each experiment.
Mentions: Diagrams of the infectious clones used in this study are shown in Fig. 1 A (below). The plasmids pU3/12 (Holt and Beachy 1991) and pU3/12 ΔM-RV (Nejidat et al. 1991) were used to generate infectious transcripts of wild-type (wt) TMV and TMV that does not express a functional MP (ΔM), respectively. The plasmids pT-MfCP (MP:GFP-CP) and pTMV-M:GfusBr (MP:GFP-ΔC) (Oparka et al. 1997) contain the MP fused to GFP in which serine at amino acid position 65 is replaced by threonine (Heim et al. 1995). vRNA-MP:GFP-ΔC does not contain the CP open reading frame (see Fig. 1 A).

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