Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Lines, Viruses, and Infection Conditions
2.2. Antibodies and Reagents
2.3. Immunofluorescence, Confocal Microscopy, and Image Analysis
2.4. Flow Cytometry and Quantification of Total M55/gB Protein
2.5. Western Blot
2.6. Quantification of Virus Growth by the Plaque Assay
2.7. Cell Viability
2.8. Data Presentation and Statistical Analysis
3. Results
3.1. The Time Course of the preAC Establishment
3.2. Dynasore Inhibits Establishment of the preAC
3.3. Other Dynamin Inhibitors, but Not Clathrin Inhibitor and Cholesterol Depletion, Prevent the Establishment of the preAC
3.4. The Dynasore Treatment Affects Pericentriolar Recruitment of EE-ERC-Derived Organelles and Reorganization of the Entire Golgi Stack in the Early Phase of MCMV Infection
3.5. Dynasore Affects Localization of Entire Golgi
3.6. Dynasore Does Not Affect Late Endosomes
3.7. Dynasore Prevents AC Formation When Present Only in the Early Phase of MCMV Infection
3.8. Dynasore Treatment in the Early Phase of Infection Blocks the Synthesis of Late MCMV Proteins and Inhibits the Release of Infective Virions
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Pericentriolar Rab10 (%) * | Golgi Patterns (%) * | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Aa | Ab | B | C | |||||||
Ø | Dy | Ø | Dy | Ø | Dy | Ø | Dy | Ø | Dy | |
Mock | 0 | - | 67.8 ± 4.2 | - | 27.2 ± 1.4 | - | 4.4 ± 2.3 | - | 0.4 ± 0.7 | - |
6 hpi | 33.3 ± 2.9 | 0.6 ± 1.1 | 7.3 ± 1.8 | 15.4 ± 4.9 | 25.9 ± 10.5 | 56.1 ± 7.2 | 42.7 ± 10.7 | 25.9 ± 9.0 | 23.9 ± 6.3 | 2.6 ± 2.9 |
14 hpi | 72.1 ± 3.6 | 5.4 ± 2.2 | 1.9 ± 0.7 | 0.9 ± 0.5 | 9.4 ± 3.9 | 58.7 ± 2.9 | 14.2 ± 4.2 | 35.3 ± 1.6 | 74.4 ± 7.5 | 5.0 ± 3.7 |
48 hpi | 78.9 ± 8.5 | 10.7 ± 5.8 | 0 | 2.7 ± 1.3 | 2.8 ± 0.7 | 70.5 ± 3.7 | 7.1 ± 4.2 | 22.9 ± 6.2 | 90.0 ± 5.0 | 3.8 ± 3.7 |
Inhibitor | Number of Cells | Rab10 Accumulation (%) * | M55 in the oAC (%) * | Viability * | ||||
---|---|---|---|---|---|---|---|---|
16 hpi | 40 hpi | 16 hpi | 40 hpi | 16 hpi | 40 hpi | 16 hpi | 40 hpi | |
Untreated | 4209 | 2386 | 65.5 ± 11.5 | 81.0 ± 5.9 | 0 | 56.6 ± 8.6 | 96.2 ± 3.0 | 90.5 ± 4.7 |
Dynasore (80 μM) | 3000 | 1757 | 2.2 ± 1.6 | 2.9 ± 2.5 | 0 | 0.1 ± 0.2 | 98.0 ± 2.8 | 92.5 ± 0.7 |
Dyngo 4a (200 μM) | 1006 | 649 | 2.4 ± 1.5 | 1.3 ± 0.3 | 0 | 0.3 ± 0.5 | 90.0 ± 3.2 | 85.0 ± 2.1 |
MiTMAB (20 μM) | 1547 | 935 | 30.0 ± 13.6 | 7.1 ± 3.0 | 0 | 0.3 ± 0.6 | 91.0 ± 4.0 | 81.0 ± 10.8 |
Dynole 34-2 (10 μM) | 1078 | 623 | 41.9 ± 8.4 | 17.6 ± 5.0 | 0 | 4.9 ± 1.8 | 88.5 ± 6.4 | 70.5 ± 9.1 |
Pitstop 2 (50 μM) | 1679 | 1652 | 45.8 ± 11.1 | 70.0 ± 9.4 | 0 | 10.5 ± 9.2 | 95.0 ± 2.4 | 89.0 ± 3.1 |
Methyl-β-CD (7.5 mM) | 1985 | 1194 | 52.9 ± 19.2 | 67.5 ± 0.8 | 0 | 37.9 ± 15.9 | 97.0 ± 2.1 | 89.0 ± 1.5 |
Marker of Cellular Compartment | The Ratio of Infected Cells (%) with the Accumulated Marker in Pericentriolar Membranes * | |||
---|---|---|---|---|
6 hpi | 14 hpi | |||
Untreated | Dynasore | Untreated | Dynasore | |
Early Endosomes | ||||
Rab5 | 50.1 ± 4.5 | 10.7 ± 1.9 | 71.5 ± 7.6 | 29.7 ± 1.6 |
Vps34 | 28.0 ± 3.5 | 4.0 ± 0.2 | 63.8 ± 4.1 | 8.3 ± 3.2 |
EEA1 | 20.7 ± 4.1 | 2.9 ± 0.6 | 62.4 ± 7.5 | 4.0 ± 1.3 |
Hrs | 37.2 ± 1.8 | 4.0 ± 0.1 | 67.3 ± 3.6 | 5.6 ± 2.9 |
Early Endosomes to Endosomal Recycling Compartment | ||||
Rab10 | 33.3 ± 2.9 | 0.6 ± 1.1 | 72.1 ± 3.6 | 5.4 ± 2.2 |
Endosomal Recycling Compartment | ||||
Rab11 | 64.1 ± 4.3 | 15.9 ± 3.9 | 78.6 ± 3.7 | 51.3 ± 7.5 |
Arf6 | 1.0 ± 0.5 | 0 | 67.81 ± 5.8 | 7.6 ± 2.7 |
Golgi Compartments | ||||
Grasp65 (ERGIC-cis-Golgi) | 28.0 ± 2.0 | 6.0 ± 0.5 | 71.8 ± 5.1 | 8.7 ± 2.8 |
GM130 (cis-Golgi) | 23.9 ± 13.3 | 2.6 ± 2.9 | 74.4 ± 7.5 | 5.0 ± 3.7 |
Golgin97 (trans-Golgi) | 29.1 ± 2.0 | 5.4 ± 3.1 | 69.2 ± 6.5 | 7.0 ± 0.6 |
Grasp65 (ERGIC-cis-Golgi) | 28.0 ± 2.0 | 6.0 ± 0.5 | 71.8 ± 5.1 | 8.7 ± 2.8 |
Late Endosomes | ||||
Lamp1 | 0 | 0 | 0 | 0 |
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Štimac, I.; Jug Vučko, N.; Blagojević Zagorac, G.; Marcelić, M.; Mahmutefendić Lučin, H.; Lučin, P. Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection. Life 2021, 11, 876. https://doi.org/10.3390/life11090876
Štimac I, Jug Vučko N, Blagojević Zagorac G, Marcelić M, Mahmutefendić Lučin H, Lučin P. Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection. Life. 2021; 11(9):876. https://doi.org/10.3390/life11090876
Chicago/Turabian StyleŠtimac, Igor, Natalia Jug Vučko, Gordana Blagojević Zagorac, Marina Marcelić, Hana Mahmutefendić Lučin, and Pero Lučin. 2021. "Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection" Life 11, no. 9: 876. https://doi.org/10.3390/life11090876
APA StyleŠtimac, I., Jug Vučko, N., Blagojević Zagorac, G., Marcelić, M., Mahmutefendić Lučin, H., & Lučin, P. (2021). Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection. Life, 11(9), 876. https://doi.org/10.3390/life11090876