Related Experiment Videos
Involvement of vacuolar proton ATPase in Junin virus multiplication
V Castilla1, L M Palermo, C E Coto
1Laboratorio de Virología, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Abstract:
The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.
Insights
Vacuolar-proton ATPase (V-H+ ATPase) inhibitors like bafilomycin A1 reduce Junin virus (JV) replication by blocking early entry and release. This confirms JV uses the endocytic pathway for cell entry.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Junin virus (JV) is a significant human pathogen.
- The role of vacuolar-proton ATPase (V-H+ ATPase) in viral replication is increasingly recognized.
- Understanding JV entry mechanisms is crucial for therapeutic development.
Purpose of the Study:
- To investigate the role of V-H+ ATPase in Junin virus replication.
- To determine the stage of the JV multiplication cycle affected by V-H+ ATPase inhibitors.
- To confirm the endocytic pathway involvement in JV cell entry.
Main Methods:
- Treatment of infected Vero and BHK-21 cells with V-H+ ATPase inhibitors (bafilomycin A1, concanamycin A).
- Assessment of viral protein expression and infectious virus production.
- Analysis of virus attachment, penetration, and release.
- Immunofluorescence staining for viral glycoproteins.
- Correlation of V-H+ ATPase activity with intracellular acidification.
Main Results:
- Bafilomycin A1 and concanamycin A significantly reduced JV replication and viral protein expression.
- Inhibitors primarily blocked early-stage JV multiplication, specifically preventing virus penetration but not attachment.
- A correlation was observed between V-H+ ATPase inhibition, reduced intracellular acidification, and decreased JV yield.
- Concanamycin A also interfered with the release of infectious JV particles.
- Intracellular transport of JV glycoproteins to the cell membrane was not affected.
Conclusions:
- V-H+ ATPase activity is essential for efficient Junin virus replication.
- JV entry into host cells occurs via the endocytic pathway.
- Inhibiting V-H+ ATPase can disrupt multiple stages of the JV life cycle, including entry and particle release.