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Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Amari K, Di Donato M, Dolja VV, Heinlein M - PLoS Pathog. (2014)

Bottom Line: The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation.The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane.Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

View Article: PubMed Central - PubMed

Affiliation: Zürich-Basel Plant Science Center, Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland.

ABSTRACT
Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

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Inhibition of myosin XI-2 disrupts the normal subcellular localization of expressed 126k:GFP.A, co-expression of 126k:GFP together with RFP. The 126k:GFP accumulates in large aggregates. B, co-expression of 126k:GFP together with myosin XI-2 tails. 126k:GFP is dispersed into numerous small aggregates. C, co-expression of 126k:GFP together with myosin XI-K tails. The 126k:GFP accumulates in large aggregates similar to those seen upon expression of RFP. Similar normal aggregates are seen also upon co-expression of the 126k:GFP with either XI-F, VIII-1, VIII-2 or VIII-B tails. D-F, co-expression of the 126k:GFP together with myosin XI-2 tails. The 126k:GFP accumulates, in addition to small aggregates, in a big aggregate in the vicinity of the nucleus. A-F, proteins were expressed by co-agroinfiltration and observed at 2 dpa. Scale bars, 20 µm (A-C); 10 µm (D-F).
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ppat-1004448-g009: Inhibition of myosin XI-2 disrupts the normal subcellular localization of expressed 126k:GFP.A, co-expression of 126k:GFP together with RFP. The 126k:GFP accumulates in large aggregates. B, co-expression of 126k:GFP together with myosin XI-2 tails. 126k:GFP is dispersed into numerous small aggregates. C, co-expression of 126k:GFP together with myosin XI-K tails. The 126k:GFP accumulates in large aggregates similar to those seen upon expression of RFP. Similar normal aggregates are seen also upon co-expression of the 126k:GFP with either XI-F, VIII-1, VIII-2 or VIII-B tails. D-F, co-expression of the 126k:GFP together with myosin XI-2 tails. The 126k:GFP accumulates, in addition to small aggregates, in a big aggregate in the vicinity of the nucleus. A-F, proteins were expressed by co-agroinfiltration and observed at 2 dpa. Scale bars, 20 µm (A-C); 10 µm (D-F).

Mentions: TMV encodes two replicase subunits, the 183 kDa (183k) and the 126 kDa (126k) proteins. The larger subunit is produced by read-through of an amber stop codon that terminates the translation of the shorter protein and harbors the RNA-dependent RNA polymerase (RdRp) domain required for replication. Although the 183k subunit is sufficient for TMV replication, the 126k subunit increases the replication rate [45] and acts as the viral suppressor of RNA interference [46], [47]. It is not clear whether the latter subunit forms part of the motile viral replication complex (VRC). Liu et al. [12] showed that transiently expressed 126k forms intracellular inclusion bodies that, similar to VRCs, align with and move along actin filaments. These findings suggest that the 126k protein may functionally contribute to the formation and trafficking of VRCs along the ER-actin network. To investigate whether the formation of 126k-containing inclusion bodies is myosin-dependent, we studied the subcellular localization of 126k and the formation of the inclusions upon inactivation of class VIII and class XI myosins. Expression of the control RFP (Figure 9A) or of myosin XI-K tails (Figure 9C), as well as of the myosin VIII-1, VIII-2, VIII-B, or XI-F tails (Figure S2) did not significantly affect the subcellular localization of 126k protein and the number or the size of inclusions. However, expression of the myosin XI-2 tails resulted in the formation of more numerous and smaller 126k inclusion bodies (Figure 9B), and also caused aggregation of 126k:GFP near the nucleus (Figure 9D-F). This perinuclear localization of 126k:GFP in the presence of myosin XI-2 tails was similar to the localization of MP seen under the same conditions (Figure 4C). Indeed, upon the expression of myosin XI-2 tails or myosin XI-K tails, the two proteins colocalize around the nucleus (Figure S3A-C) and in smaller aggregates, respectively (Figure S3D-F). This outcome is consistent with the role of myosins XI-2 and XI-K in the formation and dynamic behavior of the native ER structure (Figure 5) and suggests that the 126k protein is associated with the ER-actin network like MP. Consistently, expression of myosin XI-2 tails impaired the movement of the 126k-containing inclusion bodies along actin filaments (Figure S4). The observation of smaller 126k aggregates in the presence of myosin XI-2 tails (Figure 9B) correlates with the decrease in viral accumulation seen under the same conditions (Figure 3), and is consistent with previous reports indicating that smaller sizes of 126k-containing inclusion bodies are associated with a decrease in virus replication and with weak silencing suppression activity [12], [46]. Thus, our results support specific contributions of myosin XI-2 to the formation of larger ER-associated 126k complexes required for optimal RNA replication and the suppression of the antiviral RNA interference response.


Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Amari K, Di Donato M, Dolja VV, Heinlein M - PLoS Pathog. (2014)

Inhibition of myosin XI-2 disrupts the normal subcellular localization of expressed 126k:GFP.A, co-expression of 126k:GFP together with RFP. The 126k:GFP accumulates in large aggregates. B, co-expression of 126k:GFP together with myosin XI-2 tails. 126k:GFP is dispersed into numerous small aggregates. C, co-expression of 126k:GFP together with myosin XI-K tails. The 126k:GFP accumulates in large aggregates similar to those seen upon expression of RFP. Similar normal aggregates are seen also upon co-expression of the 126k:GFP with either XI-F, VIII-1, VIII-2 or VIII-B tails. D-F, co-expression of the 126k:GFP together with myosin XI-2 tails. The 126k:GFP accumulates, in addition to small aggregates, in a big aggregate in the vicinity of the nucleus. A-F, proteins were expressed by co-agroinfiltration and observed at 2 dpa. Scale bars, 20 µm (A-C); 10 µm (D-F).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199776&req=5

ppat-1004448-g009: Inhibition of myosin XI-2 disrupts the normal subcellular localization of expressed 126k:GFP.A, co-expression of 126k:GFP together with RFP. The 126k:GFP accumulates in large aggregates. B, co-expression of 126k:GFP together with myosin XI-2 tails. 126k:GFP is dispersed into numerous small aggregates. C, co-expression of 126k:GFP together with myosin XI-K tails. The 126k:GFP accumulates in large aggregates similar to those seen upon expression of RFP. Similar normal aggregates are seen also upon co-expression of the 126k:GFP with either XI-F, VIII-1, VIII-2 or VIII-B tails. D-F, co-expression of the 126k:GFP together with myosin XI-2 tails. The 126k:GFP accumulates, in addition to small aggregates, in a big aggregate in the vicinity of the nucleus. A-F, proteins were expressed by co-agroinfiltration and observed at 2 dpa. Scale bars, 20 µm (A-C); 10 µm (D-F).
Mentions: TMV encodes two replicase subunits, the 183 kDa (183k) and the 126 kDa (126k) proteins. The larger subunit is produced by read-through of an amber stop codon that terminates the translation of the shorter protein and harbors the RNA-dependent RNA polymerase (RdRp) domain required for replication. Although the 183k subunit is sufficient for TMV replication, the 126k subunit increases the replication rate [45] and acts as the viral suppressor of RNA interference [46], [47]. It is not clear whether the latter subunit forms part of the motile viral replication complex (VRC). Liu et al. [12] showed that transiently expressed 126k forms intracellular inclusion bodies that, similar to VRCs, align with and move along actin filaments. These findings suggest that the 126k protein may functionally contribute to the formation and trafficking of VRCs along the ER-actin network. To investigate whether the formation of 126k-containing inclusion bodies is myosin-dependent, we studied the subcellular localization of 126k and the formation of the inclusions upon inactivation of class VIII and class XI myosins. Expression of the control RFP (Figure 9A) or of myosin XI-K tails (Figure 9C), as well as of the myosin VIII-1, VIII-2, VIII-B, or XI-F tails (Figure S2) did not significantly affect the subcellular localization of 126k protein and the number or the size of inclusions. However, expression of the myosin XI-2 tails resulted in the formation of more numerous and smaller 126k inclusion bodies (Figure 9B), and also caused aggregation of 126k:GFP near the nucleus (Figure 9D-F). This perinuclear localization of 126k:GFP in the presence of myosin XI-2 tails was similar to the localization of MP seen under the same conditions (Figure 4C). Indeed, upon the expression of myosin XI-2 tails or myosin XI-K tails, the two proteins colocalize around the nucleus (Figure S3A-C) and in smaller aggregates, respectively (Figure S3D-F). This outcome is consistent with the role of myosins XI-2 and XI-K in the formation and dynamic behavior of the native ER structure (Figure 5) and suggests that the 126k protein is associated with the ER-actin network like MP. Consistently, expression of myosin XI-2 tails impaired the movement of the 126k-containing inclusion bodies along actin filaments (Figure S4). The observation of smaller 126k aggregates in the presence of myosin XI-2 tails (Figure 9B) correlates with the decrease in viral accumulation seen under the same conditions (Figure 3), and is consistent with previous reports indicating that smaller sizes of 126k-containing inclusion bodies are associated with a decrease in virus replication and with weak silencing suppression activity [12], [46]. Thus, our results support specific contributions of myosin XI-2 to the formation of larger ER-associated 126k complexes required for optimal RNA replication and the suppression of the antiviral RNA interference response.

Bottom Line: The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation.The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane.Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

View Article: PubMed Central - PubMed

Affiliation: Zürich-Basel Plant Science Center, Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland.

ABSTRACT
Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

Show MeSH
Related in: MedlinePlus