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Nuclear herpesvirus capsid motility is not dependent on F-actin.

Bosse JB, Virding S, Thiberge SY, Scherer J, Wodrich H, Ruzsics Z, Koszinowski UH, Enquist LW - MBio (2014)

Bottom Line: However, the conclusions from these studies were indirect.Moreover, in these cells, three F-actin-inhibiting drugs failed to effect capsid motility.We revisited this phenomenon and found no evidence that nuclear capsid motility depended on F-actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.

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Related in: MedlinePlus

Latrunculin A-induced actin rods bind the major capsid protein. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (mock) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (LatA). Cells were lysed, and the precleared lysates were incubated with antibodies directed against glutathione S-transferase (GST) or GFP, detecting Lifeact-EGFP (LA-GFP) and protein A/G beads for 1.5 h at 4°C. Bead-bound immunoprecipitates were separated from supernatant and washed extensively. Ten percent of lysate (input) and supernatants (sup) and 50% of immunoprecipitated protein (pellet) were subjected to SDS-PAGE and Western blotting using antiactin, anti-GFP, and anti-VP5 antibodies.
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fig5: Latrunculin A-induced actin rods bind the major capsid protein. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (mock) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (LatA). Cells were lysed, and the precleared lysates were incubated with antibodies directed against glutathione S-transferase (GST) or GFP, detecting Lifeact-EGFP (LA-GFP) and protein A/G beads for 1.5 h at 4°C. Bead-bound immunoprecipitates were separated from supernatant and washed extensively. Ten percent of lysate (input) and supernatants (sup) and 50% of immunoprecipitated protein (pellet) were subjected to SDS-PAGE and Western blotting using antiactin, anti-GFP, and anti-VP5 antibodies.

Mentions: As the previous result suggested a direct binding of nuclear capsids to actin rods, we next sought to show this interaction biochemically. To this end, we infected MEF-LA with capsid-tagged PRV and treated with LatA as described above. We then lysed the cells and used an anti-GFP antibody to immunoprecipitate Lifeact-GFP and associated proteins. By Western blotting, we detected Lifeact-GFP and actin in the immunoprecipitates of all conditions. However, only in the LatA-treated cells did we detect the major capsid protein VP5 (Fig. 5). Thus, we conclude that LatA-induced actin rods most likely trap intranuclear capsids by binding them, leading to the observed drop in motility by live-cell microscopy. This provides an intriguing alternative explanation of how LatA leads to intranuclear capsid stalling and provides a good model for why LatA influences capsid motility but the other tested F-actin-depolymerizing drugs do not.


Nuclear herpesvirus capsid motility is not dependent on F-actin.

Bosse JB, Virding S, Thiberge SY, Scherer J, Wodrich H, Ruzsics Z, Koszinowski UH, Enquist LW - MBio (2014)

Latrunculin A-induced actin rods bind the major capsid protein. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (mock) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (LatA). Cells were lysed, and the precleared lysates were incubated with antibodies directed against glutathione S-transferase (GST) or GFP, detecting Lifeact-EGFP (LA-GFP) and protein A/G beads for 1.5 h at 4°C. Bead-bound immunoprecipitates were separated from supernatant and washed extensively. Ten percent of lysate (input) and supernatants (sup) and 50% of immunoprecipitated protein (pellet) were subjected to SDS-PAGE and Western blotting using antiactin, anti-GFP, and anti-VP5 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196236&req=5

fig5: Latrunculin A-induced actin rods bind the major capsid protein. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (mock) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (LatA). Cells were lysed, and the precleared lysates were incubated with antibodies directed against glutathione S-transferase (GST) or GFP, detecting Lifeact-EGFP (LA-GFP) and protein A/G beads for 1.5 h at 4°C. Bead-bound immunoprecipitates were separated from supernatant and washed extensively. Ten percent of lysate (input) and supernatants (sup) and 50% of immunoprecipitated protein (pellet) were subjected to SDS-PAGE and Western blotting using antiactin, anti-GFP, and anti-VP5 antibodies.
Mentions: As the previous result suggested a direct binding of nuclear capsids to actin rods, we next sought to show this interaction biochemically. To this end, we infected MEF-LA with capsid-tagged PRV and treated with LatA as described above. We then lysed the cells and used an anti-GFP antibody to immunoprecipitate Lifeact-GFP and associated proteins. By Western blotting, we detected Lifeact-GFP and actin in the immunoprecipitates of all conditions. However, only in the LatA-treated cells did we detect the major capsid protein VP5 (Fig. 5). Thus, we conclude that LatA-induced actin rods most likely trap intranuclear capsids by binding them, leading to the observed drop in motility by live-cell microscopy. This provides an intriguing alternative explanation of how LatA leads to intranuclear capsid stalling and provides a good model for why LatA influences capsid motility but the other tested F-actin-depolymerizing drugs do not.

Bottom Line: However, the conclusions from these studies were indirect.Moreover, in these cells, three F-actin-inhibiting drugs failed to effect capsid motility.We revisited this phenomenon and found no evidence that nuclear capsid motility depended on F-actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.

Show MeSH
Related in: MedlinePlus