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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 viral protein distribution. MDCK cells were infected with MV at an MOI of 10. To prevent fusion, an inhibitory peptide (FIP) was added. Inhibitors (CD, Jaspla) were added at 12 h p.i.. (A) For N and M protein co-staining, cells were fixed and permeabilized with methanol/acetone. N was detected by a polyclonal rabbit antiserum and AF488-conjugated secondary antibodies. M was stained using a monoclonal antibody (MAB8910) and AF568-conjugated secondary antibodies. (B) For H and M co-staining, cells were fixed with PFA, permeabilized with Triton X-100 and H was detected with a monoclonal antibody (K83) and AF488-conjugated secondary antibodies. Afterwards, the M protein was stained with an AF555-labelled monoclonal anti-M antibody (MAB8910). xy sections of the merged images (merge xy) and a side view (xz) are shown. Images were recorded with a confocal laser scanning microscope (Zeiss LSM510). Magnification 630x.
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Figure 4: Effect of F-actin disruption and stabilization on viral protein distribution. MDCK cells were infected with MV at an MOI of 10. To prevent fusion, an inhibitory peptide (FIP) was added. Inhibitors (CD, Jaspla) were added at 12 h p.i.. (A) For N and M protein co-staining, cells were fixed and permeabilized with methanol/acetone. N was detected by a polyclonal rabbit antiserum and AF488-conjugated secondary antibodies. M was stained using a monoclonal antibody (MAB8910) and AF568-conjugated secondary antibodies. (B) For H and M co-staining, cells were fixed with PFA, permeabilized with Triton X-100 and H was detected with a monoclonal antibody (K83) and AF488-conjugated secondary antibodies. Afterwards, the M protein was stained with an AF555-labelled monoclonal anti-M antibody (MAB8910). xy sections of the merged images (merge xy) and a side view (xz) are shown. Images were recorded with a confocal laser scanning microscope (Zeiss LSM510). Magnification 630x.

Mentions: To further elucidate which assembly step is inhibited by actin disruption or stabilization, we analyzed the influence of inhibitor treatment on the intracellular localization of the viral proteins. For this purpose, co-immunostaining of M and H protein or M and N protein was performed. At 48 h p.i., cells were fixed and permeabilized and the proteins were detected with specific primary antibodies and AF488- (N protein) or AF568-labelled (M protein) secondary antibodies. As shown in Figure 4A, M and N proteins (RNPs) colocalized almost completely in control and inhibitor-treated cells. However, while M-RNP complexes in control and Jaspla-treated cells were predominantly located at the plasma membrane, the complexes accumulated in the cytoplasm after CD treatment. The intracellular localization of the proteins was confirmed by altering the gain in the confocal image to identify the cell limits. To corroborate the lack of M transport to the cell surface, M-H co-staining was performed. For this, cells were fixed and permeabilized with Triton X-100 and the H protein was detected by an H-specific antibody and AF488-conjugated secondary antibodies. The M protein was detected with an AF555-labelled monoclonal anti-M antibody. Supporting the idea of a defective M-RNP transport upon CD treatment, H and M colocalized markedly in control and Jaspla-treated cells whereas M was found in large intracellular patches in CD-treated cells (Figure 4B). The lack of H and M colocalization is clearly seen in the in the side view (vertical xz section) of the merged image. This result indicates that intact actin filaments are essentially required for M-RNP surface transport. Reduced virus release upon CD treatment is therefore concluded to be due to a defective budding as a result of a reduced amount of viral M-RNP complexes present at the plasma membrane. Interestingly, the reduced particle release upon Jaspla treatment does not appear to be linked to a defective M-RNP transport to the plasma membrane.


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 viral protein distribution. MDCK cells were infected with MV at an MOI of 10. To prevent fusion, an inhibitory peptide (FIP) was added. Inhibitors (CD, Jaspla) were added at 12 h p.i.. (A) For N and M protein co-staining, cells were fixed and permeabilized with methanol/acetone. N was detected by a polyclonal rabbit antiserum and AF488-conjugated secondary antibodies. M was stained using a monoclonal antibody (MAB8910) and AF568-conjugated secondary antibodies. (B) For H and M co-staining, cells were fixed with PFA, permeabilized with Triton X-100 and H was detected with a monoclonal antibody (K83) and AF488-conjugated secondary antibodies. Afterwards, the M protein was stained with an AF555-labelled monoclonal anti-M antibody (MAB8910). xy sections of the merged images (merge xy) and a side view (xz) are shown. Images were recorded with a confocal laser scanning microscope (Zeiss LSM510). Magnification 630x.
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Related In: Results  -  Collection

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Figure 4: Effect of F-actin disruption and stabilization on viral protein distribution. MDCK cells were infected with MV at an MOI of 10. To prevent fusion, an inhibitory peptide (FIP) was added. Inhibitors (CD, Jaspla) were added at 12 h p.i.. (A) For N and M protein co-staining, cells were fixed and permeabilized with methanol/acetone. N was detected by a polyclonal rabbit antiserum and AF488-conjugated secondary antibodies. M was stained using a monoclonal antibody (MAB8910) and AF568-conjugated secondary antibodies. (B) For H and M co-staining, cells were fixed with PFA, permeabilized with Triton X-100 and H was detected with a monoclonal antibody (K83) and AF488-conjugated secondary antibodies. Afterwards, the M protein was stained with an AF555-labelled monoclonal anti-M antibody (MAB8910). xy sections of the merged images (merge xy) and a side view (xz) are shown. Images were recorded with a confocal laser scanning microscope (Zeiss LSM510). Magnification 630x.
Mentions: To further elucidate which assembly step is inhibited by actin disruption or stabilization, we analyzed the influence of inhibitor treatment on the intracellular localization of the viral proteins. For this purpose, co-immunostaining of M and H protein or M and N protein was performed. At 48 h p.i., cells were fixed and permeabilized and the proteins were detected with specific primary antibodies and AF488- (N protein) or AF568-labelled (M protein) secondary antibodies. As shown in Figure 4A, M and N proteins (RNPs) colocalized almost completely in control and inhibitor-treated cells. However, while M-RNP complexes in control and Jaspla-treated cells were predominantly located at the plasma membrane, the complexes accumulated in the cytoplasm after CD treatment. The intracellular localization of the proteins was confirmed by altering the gain in the confocal image to identify the cell limits. To corroborate the lack of M transport to the cell surface, M-H co-staining was performed. For this, cells were fixed and permeabilized with Triton X-100 and the H protein was detected by an H-specific antibody and AF488-conjugated secondary antibodies. The M protein was detected with an AF555-labelled monoclonal anti-M antibody. Supporting the idea of a defective M-RNP transport upon CD treatment, H and M colocalized markedly in control and Jaspla-treated cells whereas M was found in large intracellular patches in CD-treated cells (Figure 4B). The lack of H and M colocalization is clearly seen in the in the side view (vertical xz section) of the merged image. This result indicates that intact actin filaments are essentially required for M-RNP surface transport. Reduced virus release upon CD treatment is therefore concluded to be due to a defective budding as a result of a reduced amount of viral M-RNP complexes present at the plasma membrane. Interestingly, the reduced particle release upon Jaspla treatment does not appear to be linked to a defective M-RNP transport to the plasma membrane.

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