Limits...
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.

Show MeSH

Related in: MedlinePlus

Influence of cytochalasin D treatment on MV glycoprotein-mediated cell-to-cell fusion. (A) MDCK cells were infected with MV at an MOI of 10 in the presence of a fusion-inhibitory peptide (FIP). CD was added at 12 h p.i., and cells were washed, detached and mixed with non-infected and non-inhibitor-treated Vero cells at 48 h p.i.. 5 h later, syncytium formation was visualized by Giemsa staining. (B) Cells were cotransfected in the presence of FIP with plasmids encoding the MV glycoproteins H and F. CD was added at 6 h post transfection, and cells were washed and detached at 24 h post transfection. 5 h after mixing with non-treated Vero cells, syncytia formation was visualized by Giemsa staining. In the upper panels, a syncytium is exemplarily shown. Fusion was quantified as described in the methods and is shown in the lower panels. Error bars indicate standard error of the mean (SEM). ** P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750272&req=5

Figure 5: Influence of cytochalasin D treatment on MV glycoprotein-mediated cell-to-cell fusion. (A) MDCK cells were infected with MV at an MOI of 10 in the presence of a fusion-inhibitory peptide (FIP). CD was added at 12 h p.i., and cells were washed, detached and mixed with non-infected and non-inhibitor-treated Vero cells at 48 h p.i.. 5 h later, syncytium formation was visualized by Giemsa staining. (B) Cells were cotransfected in the presence of FIP with plasmids encoding the MV glycoproteins H and F. CD was added at 6 h post transfection, and cells were washed and detached at 24 h post transfection. 5 h after mixing with non-treated Vero cells, syncytia formation was visualized by Giemsa staining. In the upper panels, a syncytium is exemplarily shown. Fusion was quantified as described in the methods and is shown in the lower panels. Error bars indicate standard error of the mean (SEM). ** P < 0.01.

Mentions: Actin disruption did not influence surface expression of the viral glycoproteins but led to retention of M-RNP complexes in the cytoplasm. As it is known that M downregulates cell-to-cell fusion by interacting with the cytoplasmic tails of the viral glycoproteins at the plasma membrane [1-3], we wanted to assess if syncytium formation of MV-infected MDCK cells is affected by the inhibitor treatment. Since MDCK cells do not fuse rapidly and fusion progression might be further hindered by cell-morphological alterations due to the inhibitor treatment, fusion capacity was monitored by detaching the infected, inhibitor-treated MDCK cells with accutase and subsequent mixing with a Vero cell suspension. Non-infected and non-inhibitor treated Vero cells serve here as “fusion indicator cells”. Cell mixtures were co-cultured for 5 h and syncytia formation was visualized by Giemsa staining. As shown in Figure 5A, MV-infected cells showed an increased fusion activity after CD treatment. This clearly supports our idea that CD-mediated retention of the M protein in the cytoplasm resulted in a reduced M-glycoprotein interaction at the plasma membrane, and thus to a reduced fusion downregulation by M. Since it has been proposed that actin structures can also restrict fusion-pore extension [16,17], we wanted to rule out that the observed increased syncytia formation upon CD treatment might be simply due to the disruption of the actin cytocortex facilitating expansion of the fusion pores. We therefore analyzed the effect of CD treatment on MV glycoprotein-mediated cell-to-cell fusion in the absence of M and any virus infection. For this purpose, we coexpressed H and F proteins in MDCK cells, incubated the cells either in the absence or presence of CD for 18 h, and performed the fusion assay as described above. In contrast to the infection, H and F cotransfected cells did not show enhanced fusion capacity after CD-mediated actin disruption. We thus conclude that the increase of the relative fusion after CD treatment in MV-infected cells is due to a reduced M-mediated fusion downregulation, and is therefore the consequence of the defective M surface transport in cells with a disrupted actin cytoskeleton.


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

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

Influence of cytochalasin D treatment on MV glycoprotein-mediated cell-to-cell fusion. (A) MDCK cells were infected with MV at an MOI of 10 in the presence of a fusion-inhibitory peptide (FIP). CD was added at 12 h p.i., and cells were washed, detached and mixed with non-infected and non-inhibitor-treated Vero cells at 48 h p.i.. 5 h later, syncytium formation was visualized by Giemsa staining. (B) Cells were cotransfected in the presence of FIP with plasmids encoding the MV glycoproteins H and F. CD was added at 6 h post transfection, and cells were washed and detached at 24 h post transfection. 5 h after mixing with non-treated Vero cells, syncytia formation was visualized by Giemsa staining. In the upper panels, a syncytium is exemplarily shown. Fusion was quantified as described in the methods and is shown in the lower panels. Error bars indicate standard error of the mean (SEM). ** P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Influence of cytochalasin D treatment on MV glycoprotein-mediated cell-to-cell fusion. (A) MDCK cells were infected with MV at an MOI of 10 in the presence of a fusion-inhibitory peptide (FIP). CD was added at 12 h p.i., and cells were washed, detached and mixed with non-infected and non-inhibitor-treated Vero cells at 48 h p.i.. 5 h later, syncytium formation was visualized by Giemsa staining. (B) Cells were cotransfected in the presence of FIP with plasmids encoding the MV glycoproteins H and F. CD was added at 6 h post transfection, and cells were washed and detached at 24 h post transfection. 5 h after mixing with non-treated Vero cells, syncytia formation was visualized by Giemsa staining. In the upper panels, a syncytium is exemplarily shown. Fusion was quantified as described in the methods and is shown in the lower panels. Error bars indicate standard error of the mean (SEM). ** P < 0.01.
Mentions: Actin disruption did not influence surface expression of the viral glycoproteins but led to retention of M-RNP complexes in the cytoplasm. As it is known that M downregulates cell-to-cell fusion by interacting with the cytoplasmic tails of the viral glycoproteins at the plasma membrane [1-3], we wanted to assess if syncytium formation of MV-infected MDCK cells is affected by the inhibitor treatment. Since MDCK cells do not fuse rapidly and fusion progression might be further hindered by cell-morphological alterations due to the inhibitor treatment, fusion capacity was monitored by detaching the infected, inhibitor-treated MDCK cells with accutase and subsequent mixing with a Vero cell suspension. Non-infected and non-inhibitor treated Vero cells serve here as “fusion indicator cells”. Cell mixtures were co-cultured for 5 h and syncytia formation was visualized by Giemsa staining. As shown in Figure 5A, MV-infected cells showed an increased fusion activity after CD treatment. This clearly supports our idea that CD-mediated retention of the M protein in the cytoplasm resulted in a reduced M-glycoprotein interaction at the plasma membrane, and thus to a reduced fusion downregulation by M. Since it has been proposed that actin structures can also restrict fusion-pore extension [16,17], we wanted to rule out that the observed increased syncytia formation upon CD treatment might be simply due to the disruption of the actin cytocortex facilitating expansion of the fusion pores. We therefore analyzed the effect of CD treatment on MV glycoprotein-mediated cell-to-cell fusion in the absence of M and any virus infection. For this purpose, we coexpressed H and F proteins in MDCK cells, incubated the cells either in the absence or presence of CD for 18 h, and performed the fusion assay as described above. In contrast to the infection, H and F cotransfected cells did not show enhanced fusion capacity after CD-mediated actin disruption. We thus conclude that the increase of the relative fusion after CD treatment in MV-infected cells is due to a reduced M-mediated fusion downregulation, and is therefore the consequence of the defective M surface transport in cells with a disrupted actin cytoskeleton.

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