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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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Viral protein expression upon F-actin disruption and stabilization. MDCK cells were infected with MV at an MOI of 10 (control, CD, Jaspla), or left uninfected (mock). Inhibitors were added at 12 h p.i.. At 48 h p.i., cells were surface biotinylated and subsequently lysed in RIPA buffer. (A) To determine the amount of MV glycoproteins expressed on the cell surface, H and F were immunprecipitated from cell lysates with specific monoclonal antibodies. Samples were then separated by SDS-PAGE, transferred to nitrocellulose and probed with AF680-conjugated streptavidin. (B) M protein was immunoprecipitated from cell lysates using an anti-M specific antibody. Precipitates were subjected to western blot analysis using M-specific primary antibodies and AF680-conjugated secondary antibodies. (C) For N, tubulin and actin staining, lysates were directly subjected to SDS-PAGE, blotted to nitrocelluose and incubated with specific primary antibodies and fluorophore-conjugated secondary antibodies. Blots were scanned with a Li-Cor Odyssey IR system.
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Figure 3: Viral protein expression upon F-actin disruption and stabilization. MDCK cells were infected with MV at an MOI of 10 (control, CD, Jaspla), or left uninfected (mock). Inhibitors were added at 12 h p.i.. At 48 h p.i., cells were surface biotinylated and subsequently lysed in RIPA buffer. (A) To determine the amount of MV glycoproteins expressed on the cell surface, H and F were immunprecipitated from cell lysates with specific monoclonal antibodies. Samples were then separated by SDS-PAGE, transferred to nitrocellulose and probed with AF680-conjugated streptavidin. (B) M protein was immunoprecipitated from cell lysates using an anti-M specific antibody. Precipitates were subjected to western blot analysis using M-specific primary antibodies and AF680-conjugated secondary antibodies. (C) For N, tubulin and actin staining, lysates were directly subjected to SDS-PAGE, blotted to nitrocelluose and incubated with specific primary antibodies and fluorophore-conjugated secondary antibodies. Blots were scanned with a Li-Cor Odyssey IR system.

Mentions: To determine if the reduced virus release upon CD and Jaspla treatment is due to an overall change in viral protein expression levels or an altered surface expression of the viral glycoproteins, MV-infected and inhibitor-treated cells were surface biotinylated. For this purpose, the cell membrane impermeant reagent S-NHS-biotin was added at 48 h p.i., the time point at which control virus release was almost maximal and the inhibitor-induced reduction of virus release was most pronounced. After biotin labelling, cells were lysed and the MV glycoproteins H and F were immunoprecipitated by specific antibodies. Precipitates were separated by SDS-PAGE and transferred to nitrocellulose. Biotinylated H and F proteins were then detected using AF680-conjugated streptavidin (Figure 3A). Since the viral M protein is an intracellular protein and thus not surface biotinylated, it was detected after immunoprecipitation by Western blot analysis (Figure 3B). The viral N protein and cellular tubulin and actin were directly detected in the cell lysates by western blot analysis (Figure 3C). Figure 3 shows no substantial differences in the protein amounts in inhibitor-treated cells compared to control cells. We thus conclude that neither changes in the overall protein expression levels, nor a decreased surface expression of the viral glycoproteins can be responsible for the observed reduction in virus particle formation upon actin filament disruption or stabilization.


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

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

Viral protein expression upon F-actin disruption and stabilization. MDCK cells were infected with MV at an MOI of 10 (control, CD, Jaspla), or left uninfected (mock). Inhibitors were added at 12 h p.i.. At 48 h p.i., cells were surface biotinylated and subsequently lysed in RIPA buffer. (A) To determine the amount of MV glycoproteins expressed on the cell surface, H and F were immunprecipitated from cell lysates with specific monoclonal antibodies. Samples were then separated by SDS-PAGE, transferred to nitrocellulose and probed with AF680-conjugated streptavidin. (B) M protein was immunoprecipitated from cell lysates using an anti-M specific antibody. Precipitates were subjected to western blot analysis using M-specific primary antibodies and AF680-conjugated secondary antibodies. (C) For N, tubulin and actin staining, lysates were directly subjected to SDS-PAGE, blotted to nitrocelluose and incubated with specific primary antibodies and fluorophore-conjugated secondary antibodies. Blots were scanned with a Li-Cor Odyssey IR system.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Viral protein expression upon F-actin disruption and stabilization. MDCK cells were infected with MV at an MOI of 10 (control, CD, Jaspla), or left uninfected (mock). Inhibitors were added at 12 h p.i.. At 48 h p.i., cells were surface biotinylated and subsequently lysed in RIPA buffer. (A) To determine the amount of MV glycoproteins expressed on the cell surface, H and F were immunprecipitated from cell lysates with specific monoclonal antibodies. Samples were then separated by SDS-PAGE, transferred to nitrocellulose and probed with AF680-conjugated streptavidin. (B) M protein was immunoprecipitated from cell lysates using an anti-M specific antibody. Precipitates were subjected to western blot analysis using M-specific primary antibodies and AF680-conjugated secondary antibodies. (C) For N, tubulin and actin staining, lysates were directly subjected to SDS-PAGE, blotted to nitrocelluose and incubated with specific primary antibodies and fluorophore-conjugated secondary antibodies. Blots were scanned with a Li-Cor Odyssey IR system.
Mentions: To determine if the reduced virus release upon CD and Jaspla treatment is due to an overall change in viral protein expression levels or an altered surface expression of the viral glycoproteins, MV-infected and inhibitor-treated cells were surface biotinylated. For this purpose, the cell membrane impermeant reagent S-NHS-biotin was added at 48 h p.i., the time point at which control virus release was almost maximal and the inhibitor-induced reduction of virus release was most pronounced. After biotin labelling, cells were lysed and the MV glycoproteins H and F were immunoprecipitated by specific antibodies. Precipitates were separated by SDS-PAGE and transferred to nitrocellulose. Biotinylated H and F proteins were then detected using AF680-conjugated streptavidin (Figure 3A). Since the viral M protein is an intracellular protein and thus not surface biotinylated, it was detected after immunoprecipitation by Western blot analysis (Figure 3B). The viral N protein and cellular tubulin and actin were directly detected in the cell lysates by western blot analysis (Figure 3C). Figure 3 shows no substantial differences in the protein amounts in inhibitor-treated cells compared to control cells. We thus conclude that neither changes in the overall protein expression levels, nor a decreased surface expression of the viral glycoproteins can be responsible for the observed reduction in virus particle formation upon actin filament disruption or stabilization.

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