scholarly journals Replication of Tobacco Mosaic Virus on Endoplasmic Reticulum and Role of the Cytoskeleton and Virus Movement Protein in Intracellular Distribution of Viral RNA

1999 ◽  
Vol 147 (5) ◽  
pp. 945-958 ◽  
Author(s):  
Paloma Más ◽  
Roger N. Beachy
Keyword(s):  
In Situ Hybridization ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Viral Rna ◽  
Pharmacological Agents ◽  
Intracellular Targeting ◽  
Cytoplasmic Filaments ◽  
Infected Cells

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.

scholarly journals A Dysfunctional Movement Protein of Tobacco mosaic virus Interferes with Targeting of Wild-Type Movement Protein to Microtubules

2001 ◽  
Vol 14 (7) ◽  
pp. 895-904 ◽  
Author(s):  
Guy Kotlizky ◽  
Aviva Katz ◽  
Jessica van der Laak ◽  
Vitaly Boyko ◽  
Moshe Lapidot ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Free System ◽  
Intracellular Targeting ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Dependent Processes ◽  
Cell Spread

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.

scholarly journals Challenging the Role of Microtubules in Tobacco Mosaic Virus Movement by Drug Treatments Is Disputable

2006 ◽  
Vol 80 (13) ◽  
pp. 6712-6715 ◽  
Author(s):  
Mark Seemanpillai ◽  
Rabab Elamawi ◽  
Christophe Ritzenthaler ◽  
Manfred Heinlein
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Viral Rna ◽  
Virus Movement ◽  
Direct Role ◽  
Microtubule Inhibitors ◽  
Microtubule Polymerization ◽  
Drug Treatments

ABSTRACT The movement protein (MP) of Tobacco mosaic virus interacts with microtubules during infection. Although this interaction is correlated with the function of MP in the cell-to-cell transport of viral RNA, a direct role of microtubules in the movement process was recently challenged by studies involving the treatment of plants with inhibitors of microtubule polymerization. Here, we report evidence suggesting that such treatments may not efficiently disrupt all microtubules. Thus, results obtained from studies using microtubule inhibitors may have to remain open to interpretation with regard to the involvement of microtubules in viral RNA trafficking.

scholarly journals In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats

1998 ◽  
Vol 88 (10) ◽  
pp. 1031-1039 ◽  
Author(s):  
Petra H. Nass ◽  
Leslie L. Domier ◽  
Birute P. Jakstys ◽  
Cleora J. D'Arcy
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Barley Yellow Dwarf Virus ◽  
Yellow Dwarf ◽  
Viral Rna ◽  
Barley Yellow Dwarf ◽  
Infected Cells ◽  
Yellow Dwarf Virus ◽  
Filamentous Material

Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

scholarly journals Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

2020 ◽  
Vol 8 (12) ◽  
pp. 1928
Author(s):  
Sami Salmikangas ◽  
Jutta E. Laiho ◽  
Kerttu Kalander ◽  
Mira Laajala ◽  
Anni Honkimaa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Viral Rna ◽  
Quantitative Real Time Pcr ◽  
Virion Assembly ◽  
Rna Molecules ◽  
Branched Dna ◽  
Infected Cells ◽  
Good Signal

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.

Movement protein stimulates tobacco mosaic virus reproduction in infected cells

2006 ◽  
Vol 409 (1) ◽  
pp. 253-256 ◽  
Author(s):  
L. G. Tyulkina ◽  
E. V. Skurat ◽  
A. S. Zvereva ◽  
Yu. L. Dorokhov ◽  
J. G. Atabekov
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Virus Reproduction ◽  
Infected Cells

scholarly journals Intramolecular Complementing Mutations in Tobacco Mosaic Virus Movement Protein Confirm a Role for Microtubule Association in Viral RNA Transport

2002 ◽  
Vol 76 (8) ◽  
pp. 3974-3980 ◽  
Author(s):  
Vitaly Boyko ◽  
Jamie Alan Ashby ◽  
Elena Suslova ◽  
Jacqueline Ferralli ◽  
Oliver Sterthaus ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Microscopic Analysis ◽  
Viral Rna ◽  
Cell Transport ◽  
Substitution Mutations ◽  
Green Fluorescent

ABSTRACT The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MPP81S-GFP and MPP81S;T104I;R167K-GFP differ in subcellular distribution. MPP81S-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MPP81S;T104I;R167K-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.

Plasmodesmata and intercellular transport of viral RNA

2007 ◽  
Vol 35 (1) ◽  
pp. 142-145 ◽  
Author(s):  
C. Hofmann ◽  
A. Sambade ◽  
M. Heinlein
Keyword(s):  
Cell Wall ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Plant Cell Wall ◽  
Movement Protein ◽  
Cell Communication ◽  
Viral Rna ◽  
Intercellular Transport ◽  
Cytoplasmic Bridges ◽  
Spread Of Infection

Cell-to-cell communication in plants involves the symplastic trafficking of informational protein and RNA macromolecules through cytoplasmic bridges in the plant cell wall known as plasmodesmata. Viruses exploit this route for the spread of infection and are used as a model to study the mechanisms by which macromolecules are targeted to the pore. Studies using tobacco mosaic virus have led to the identification of host components that participate in plasmodesmal targeting of viral RNA and movement protein.

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