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Actin filaments disruption and stabilization affect measles virus maturation by different mechanisms.

Dietzel E, Kolesnikova L, Maisner A - Virol. J. (2013)

Bottom Line: Supporting our finding that F-actin disruption blocks M-RNP transport to the plasma membrane, cell-to-cell spread of MV infection was enhanced upon CD treatment.Due to the lack of M-glycoprotein-interactions at the cell surface, M-mediated fusion downregulation was hindered and a more rapid syncytia formation was observed.While stable actin filaments are needed for intracellular trafficking of viral RNPs to the plasma membrane, and consequently for assembly at the cell surface and prevention of an overexerted fusion by the viral surface glycoproteins, actin dynamics are required for the final steps of budding at the plasma membrane.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, Philipps University of Marburg, Hans-Meerwein-Str 2, Marburg, D-35043, Germany.

ABSTRACT

Background: Cytoskeletal proteins are often involved in the virus life cycle, either at early steps during virus entry or at later steps during formation of new virus particles. Though actin filaments have been shown to play a role in the production of measles virus (MV), the importance of actin dynamics for virus assembly and budding steps is not known yet. Aim of this work was thus to analyze the distinctive consequences of F-actin stabilization or disruption for MV protein trafficking, particle assembly and virus release.

Results: MV infection studies in the presence of inhibitors differently affecting the actin cytoskeleton revealed that not only actin disruption but also stabilization of actin filaments interfered with MV particle release. While overall viral protein synthesis, surface expression levels of the MV glycoproteins, and cell-associated infectivity was not altered, cell-free virus titers were decreased. Interestingly, the underlying mechanisms of interference with late MV maturation steps differed principally after F-actin disruption by Cytochalasin D (CD) and F-actin stabilization by Jasplakinolide (Jaspla). While intact actin filaments were shown to be required for transport of nucleocapsids and matrix proteins (M-RNPs) from inclusions to the plasma membrane, actin dynamics at the cytocortex that are blocked by Jaspla are necessary for final steps in virus assembly, in particular for the formation of viral buds and the pinching-off at the plasma membrane. Supporting our finding that F-actin disruption blocks M-RNP transport to the plasma membrane, cell-to-cell spread of MV infection was enhanced upon CD treatment. Due to the lack of M-glycoprotein-interactions at the cell surface, M-mediated fusion downregulation was hindered and a more rapid syncytia formation was observed.

Conclusion: While stable actin filaments are needed for intracellular trafficking of viral RNPs to the plasma membrane, and consequently for assembly at the cell surface and prevention of an overexerted fusion by the viral surface glycoproteins, actin dynamics are required for the final steps of budding at the plasma membrane.

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Effect of F-actin stabilization on MV budding. Electron microscopic analysis of ultrathin sections of MV-infected Vero cells either left without treatment (A, control), or treated with 0.2 μM Jaspla at 12 h p.i. (B and C). White-line marked areas show accumulations of RNPs in the cytoplasm. Black-boxed areas on upper panels are shown at higher magnifications in lower panels. Black arrows indicate RNPs inside viral particles; white arrows indicate RNPs beneath the plasma membrane.
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Figure 6: Effect of F-actin stabilization on MV budding. Electron microscopic analysis of ultrathin sections of MV-infected Vero cells either left without treatment (A, control), or treated with 0.2 μM Jaspla at 12 h p.i. (B and C). White-line marked areas show accumulations of RNPs in the cytoplasm. Black-boxed areas on upper panels are shown at higher magnifications in lower panels. Black arrows indicate RNPs inside viral particles; white arrows indicate RNPs beneath the plasma membrane.

Mentions: Jaspla treatment reduced virus release by more than 80% without affecting viral protein synthesis or downregulating the surface transport of viral glycoproteins and M-RNP complexes. Thus, very late budding steps at the plasma membrane appeared to be affected by actin stabilization. To address this question, we performed ultrastructural analyses of MV-infected Vero cells, in which 200 nM Jaspla treatment resulted in reduction of virus release into the supernatant by 83% (data not shown). For the EM analysis, MV-infected cells were treated with Jaspla at 12 h p.i.. After fixation at 48 h p.i., cells were processed for EM analysis. While in control cells, virus particles and bud formation were abundantly found (Figure 6A), detection of virus buds and cell-free particles was much more restricted in Jaspla-treated cells (Figure 6B, 6C). RNPs were highly concentrated and aligned at the plasma membrane. However, budding events are rarely seen in Jaspla-treated cells (Figure 6B, 6C, lower panels). This clearly confirms that actin stabilization does not block delivery of M-RNP complexes to the plasma membrane but rather prevents the formation of viral buds and the final pinching off.


Actin filaments disruption and stabilization affect measles virus maturation by different mechanisms.

Dietzel E, Kolesnikova L, Maisner A - Virol. J. (2013)

Effect of F-actin stabilization on MV budding. Electron microscopic analysis of ultrathin sections of MV-infected Vero cells either left without treatment (A, control), or treated with 0.2 μM Jaspla at 12 h p.i. (B and C). White-line marked areas show accumulations of RNPs in the cytoplasm. Black-boxed areas on upper panels are shown at higher magnifications in lower panels. Black arrows indicate RNPs inside viral particles; white arrows indicate RNPs beneath the plasma membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750272&req=5

Figure 6: Effect of F-actin stabilization on MV budding. Electron microscopic analysis of ultrathin sections of MV-infected Vero cells either left without treatment (A, control), or treated with 0.2 μM Jaspla at 12 h p.i. (B and C). White-line marked areas show accumulations of RNPs in the cytoplasm. Black-boxed areas on upper panels are shown at higher magnifications in lower panels. Black arrows indicate RNPs inside viral particles; white arrows indicate RNPs beneath the plasma membrane.
Mentions: Jaspla treatment reduced virus release by more than 80% without affecting viral protein synthesis or downregulating the surface transport of viral glycoproteins and M-RNP complexes. Thus, very late budding steps at the plasma membrane appeared to be affected by actin stabilization. To address this question, we performed ultrastructural analyses of MV-infected Vero cells, in which 200 nM Jaspla treatment resulted in reduction of virus release into the supernatant by 83% (data not shown). For the EM analysis, MV-infected cells were treated with Jaspla at 12 h p.i.. After fixation at 48 h p.i., cells were processed for EM analysis. While in control cells, virus particles and bud formation were abundantly found (Figure 6A), detection of virus buds and cell-free particles was much more restricted in Jaspla-treated cells (Figure 6B, 6C). RNPs were highly concentrated and aligned at the plasma membrane. However, budding events are rarely seen in Jaspla-treated cells (Figure 6B, 6C, lower panels). This clearly confirms that actin stabilization does not block delivery of M-RNP complexes to the plasma membrane but rather prevents the formation of viral buds and the final pinching off.

Bottom Line: Supporting our finding that F-actin disruption blocks M-RNP transport to the plasma membrane, cell-to-cell spread of MV infection was enhanced upon CD treatment.Due to the lack of M-glycoprotein-interactions at the cell surface, M-mediated fusion downregulation was hindered and a more rapid syncytia formation was observed.While stable actin filaments are needed for intracellular trafficking of viral RNPs to the plasma membrane, and consequently for assembly at the cell surface and prevention of an overexerted fusion by the viral surface glycoproteins, actin dynamics are required for the final steps of budding at the plasma membrane.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, Philipps University of Marburg, Hans-Meerwein-Str 2, Marburg, D-35043, Germany.

ABSTRACT

Background: Cytoskeletal proteins are often involved in the virus life cycle, either at early steps during virus entry or at later steps during formation of new virus particles. Though actin filaments have been shown to play a role in the production of measles virus (MV), the importance of actin dynamics for virus assembly and budding steps is not known yet. Aim of this work was thus to analyze the distinctive consequences of F-actin stabilization or disruption for MV protein trafficking, particle assembly and virus release.

Results: MV infection studies in the presence of inhibitors differently affecting the actin cytoskeleton revealed that not only actin disruption but also stabilization of actin filaments interfered with MV particle release. While overall viral protein synthesis, surface expression levels of the MV glycoproteins, and cell-associated infectivity was not altered, cell-free virus titers were decreased. Interestingly, the underlying mechanisms of interference with late MV maturation steps differed principally after F-actin disruption by Cytochalasin D (CD) and F-actin stabilization by Jasplakinolide (Jaspla). While intact actin filaments were shown to be required for transport of nucleocapsids and matrix proteins (M-RNPs) from inclusions to the plasma membrane, actin dynamics at the cytocortex that are blocked by Jaspla are necessary for final steps in virus assembly, in particular for the formation of viral buds and the pinching-off at the plasma membrane. Supporting our finding that F-actin disruption blocks M-RNP transport to the plasma membrane, cell-to-cell spread of MV infection was enhanced upon CD treatment. Due to the lack of M-glycoprotein-interactions at the cell surface, M-mediated fusion downregulation was hindered and a more rapid syncytia formation was observed.

Conclusion: While stable actin filaments are needed for intracellular trafficking of viral RNPs to the plasma membrane, and consequently for assembly at the cell surface and prevention of an overexerted fusion by the viral surface glycoproteins, actin dynamics are required for the final steps of budding at the plasma membrane.

Show MeSH
Related in: MedlinePlus