Related Experiment Videos
Association of the caveola vesicular system with cellular entry by filoviruses
Cyril J Empig1, Mark A Goldsmith
1Gladstone Institute of Virology and Immunology, San Francisco, California 94141-9100, USA.
Abstract:
The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.
Insights
Filoviruses like Ebola and Marburg may enter human cells using caveolae, a specific cellular pathway. This study used pseudotyping to show that inhibiting caveolae blocks filovirus entry, suggesting a novel mechanism for infection.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Filoviruses, including Ebola Zaire virus and Marburg virus, are known human pathogens.
- The precise mechanism of filovirus entry into target cells remains largely uncharacterized.
- Endocytic vesicles are hypothesized to be involved in filovirus cellular uptake.
Purpose of the Study:
- To investigate the cell biology and entry pathway of filoviruses.
- To determine whether filoviruses utilize caveolae or clathrin-mediated endocytosis for cell entry.
- To utilize a pseudotyping strategy for studying filovirus entry mechanisms.
Main Methods:
- Filovirus pseudotypes were generated to mimic viral entry.
- Inhibitors of the caveola system (cholesterol-sequestering drugs, phorbol esters) were used to assess entry.
- Cell entry kinetics were compared between filovirus and Vesicular Stomatitis Virus (VSV) pseudotypes.
- Immunofluorescence and confocal microscopy were employed to visualize pseudotype localization.
Main Results:
- Inhibitors targeting the caveola system significantly reduced filovirus pseudotype entry into human cells.
- Filovirus pseudotype entry kinetics were slower compared to VSV pseudotypes, which use clathrin-mediated endocytosis.
- Confocal microscopy demonstrated colocalization of filovirus pseudotypes with caveolin-1, a marker for caveolae.
- VSV pseudotypes did not colocalize with caveolin-1, confirming pathway specificity.
Conclusions:
- The findings provide strong evidence that filoviruses utilize caveolae for cellular entry.
- This study elucidates a critical step in the filovirus infection cycle.
- Understanding filovirus entry via caveolae may inform future antiviral strategies.