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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 disruption and stabilization on virus release and cell-associated infectivity. MDCK cells were infected with MV at an MOI of 10. Inhibitors were added at 12 h p.i. Supernatants were taken in 12 hour intervals to titrate cell-free virus and cells were harvested at 24, 48 and 72 h p.i. To titrate cell-associated infectivity, virus titers were quantitated by plaque assay. Values plotted represent mean results of two independent experiments.
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Figure 2: Effect of F-actin disruption and stabilization on virus release and cell-associated infectivity. MDCK cells were infected with MV at an MOI of 10. Inhibitors were added at 12 h p.i. Supernatants were taken in 12 hour intervals to titrate cell-free virus and cells were harvested at 24, 48 and 72 h p.i. To titrate cell-associated infectivity, virus titers were quantitated by plaque assay. Values plotted represent mean results of two independent experiments.

Mentions: To analyze the importance of the actin polymerization and depolymerization on MV infection, we quantitated the release of infectious MV particles and the cell-associated infectivity in the absence and presence of 4 μM Cytochalasin D (CD) or 100 nM Jasplakinolide (Jaspla). To visualize the effect and the specificity of the two inhibitors, actin filaments and microtubules in non-infected cells treated with CD or Jaspla were either stained with Phalloidin-FITC or with an anti-tubulin antibody and an AlexaFluor (AF)488-labelled secondary antibody, respectively. Actin stress fibers and subcortical actin were readily stained in control cells (Figure 1A). As CD causes actin disruption by binding to the barbed plus ends of actin filaments, treatment with 4 μM CD led to the disappearance of stress fibers and the actin-dense cytocortex. Actin was rather found in punctate structures. In contrast to CD, the effect of the actin filament stabilizing drug Jaspla was evident by the formation of perinuclear F-actin aggresomes [15] (Figure 1A). None of the inhibitors affected the filamentous structure of microtubules (Figure 1B) demonstrating that their effect was specific for actin and not due to a general breakdown of the cell cytoskeleton. To exclude that cytotoxic effects due to inhibitor treatment affect the analyses, cell viability was tested at 48 h p.i. by incubating live cells with propidium iodide (PI), a membrane impermeant DNA dye, followed by cell permeabilization and nuclear DAPI staining. Control and inhibitor treated cells were negative for PI staining, confirming that the cells were viable and that the plasma membrane was still intact (data not shown). To monitor MV infection in the presence of the inhibitors, MDCK cells were infected with MV at an MOI of 10. At 12 h p.i., inhibitors were added and the cells were further incubated up to 72 h p.i.. Aliquots from the cell supernatants were taken every 12 h to determine the cell-free virus titers. To determine the cell-associated infectivity, cell lysates were prepared by one freeze-thaw cycle and were titrated by plaque assay on confluent Vero cells. Cell disruption by freezing and thawing allows isolation of intracellular viral RNPs not yet finally assembled or budding at the plasma membrane. Figure 2 shows that this cell-associated infectivity was not reduced by CD or Jaspla treatment suggesting that inhibitor treatments did not principally diminish the number of infected cells or the production of infectious RNPs and viral proteins. In contrast, virus titers in the supernatants were clearly reduced. In CD- and Jaspla-treated cells, virus release at 48 h p.i. was reduced by 99% (2 log steps) and 80% (1 log step), respectively. A similar reduction of virus release by CD and Jaspla treatment was observed in Vero cells (data not shown). In agreement with the described reversibility of the actin inhibitors, the effects of the inhibtors on the production of viruses were reversible. When we removed the inhibitors at 48 h p.i., final virus titers almost reached control levels within 24 h. While control cells released 5.1×105 p.f.u./ml between 48 and 72 h p.i., virus yield produced between the time of CD and Jaspla removal and 72 h p.i. increased up to 1.7×105 p.f.u./ml and 3.5×105 p.f.u./ml, respectively.


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 disruption and stabilization on virus release and cell-associated infectivity. MDCK cells were infected with MV at an MOI of 10. Inhibitors were added at 12 h p.i. Supernatants were taken in 12 hour intervals to titrate cell-free virus and cells were harvested at 24, 48 and 72 h p.i. To titrate cell-associated infectivity, virus titers were quantitated by plaque assay. Values plotted represent mean results of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of F-actin disruption and stabilization on virus release and cell-associated infectivity. MDCK cells were infected with MV at an MOI of 10. Inhibitors were added at 12 h p.i. Supernatants were taken in 12 hour intervals to titrate cell-free virus and cells were harvested at 24, 48 and 72 h p.i. To titrate cell-associated infectivity, virus titers were quantitated by plaque assay. Values plotted represent mean results of two independent experiments.
Mentions: To analyze the importance of the actin polymerization and depolymerization on MV infection, we quantitated the release of infectious MV particles and the cell-associated infectivity in the absence and presence of 4 μM Cytochalasin D (CD) or 100 nM Jasplakinolide (Jaspla). To visualize the effect and the specificity of the two inhibitors, actin filaments and microtubules in non-infected cells treated with CD or Jaspla were either stained with Phalloidin-FITC or with an anti-tubulin antibody and an AlexaFluor (AF)488-labelled secondary antibody, respectively. Actin stress fibers and subcortical actin were readily stained in control cells (Figure 1A). As CD causes actin disruption by binding to the barbed plus ends of actin filaments, treatment with 4 μM CD led to the disappearance of stress fibers and the actin-dense cytocortex. Actin was rather found in punctate structures. In contrast to CD, the effect of the actin filament stabilizing drug Jaspla was evident by the formation of perinuclear F-actin aggresomes [15] (Figure 1A). None of the inhibitors affected the filamentous structure of microtubules (Figure 1B) demonstrating that their effect was specific for actin and not due to a general breakdown of the cell cytoskeleton. To exclude that cytotoxic effects due to inhibitor treatment affect the analyses, cell viability was tested at 48 h p.i. by incubating live cells with propidium iodide (PI), a membrane impermeant DNA dye, followed by cell permeabilization and nuclear DAPI staining. Control and inhibitor treated cells were negative for PI staining, confirming that the cells were viable and that the plasma membrane was still intact (data not shown). To monitor MV infection in the presence of the inhibitors, MDCK cells were infected with MV at an MOI of 10. At 12 h p.i., inhibitors were added and the cells were further incubated up to 72 h p.i.. Aliquots from the cell supernatants were taken every 12 h to determine the cell-free virus titers. To determine the cell-associated infectivity, cell lysates were prepared by one freeze-thaw cycle and were titrated by plaque assay on confluent Vero cells. Cell disruption by freezing and thawing allows isolation of intracellular viral RNPs not yet finally assembled or budding at the plasma membrane. Figure 2 shows that this cell-associated infectivity was not reduced by CD or Jaspla treatment suggesting that inhibitor treatments did not principally diminish the number of infected cells or the production of infectious RNPs and viral proteins. In contrast, virus titers in the supernatants were clearly reduced. In CD- and Jaspla-treated cells, virus release at 48 h p.i. was reduced by 99% (2 log steps) and 80% (1 log step), respectively. A similar reduction of virus release by CD and Jaspla treatment was observed in Vero cells (data not shown). In agreement with the described reversibility of the actin inhibitors, the effects of the inhibtors on the production of viruses were reversible. When we removed the inhibitors at 48 h p.i., final virus titers almost reached control levels within 24 h. While control cells released 5.1×105 p.f.u./ml between 48 and 72 h p.i., virus yield produced between the time of CD and Jaspla removal and 72 h p.i. increased up to 1.7×105 p.f.u./ml and 3.5×105 p.f.u./ml, respectively.

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