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Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells
1Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Spain.
Abstract:
The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.
Insights
Influenza virus entry into cells requires a pH gradient, not just low pH. Inhibiting vacuolar proton-ATPase (v-[H+]ATPase) with antibiotics like bafilomycin A1 blocked viral entry, highlighting the importance of endosomal acidification.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Endosomal acidification is crucial for the entry of many viruses.
- Vacuolar proton-ATPase (v-[H+]ATPase) is the primary enzyme responsible for maintaining the acidic environment within endosomes.
Purpose of the Study:
- To investigate the specific role of endosomal acidification in influenza virus entry into Madin-Darby canine kidney (MDCK) cells.
- To determine whether low pH or a pH gradient is essential for viral entry.
Main Methods:
- Utilized selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), including bafilomycin A1 and concanamycin A.
- Assessed the effect of these inhibitors and other proton-ATPase inhibitors (N,N'-dicyclohexylcarbodiimide, elaiophylin) on influenza virus entry.
- Examined the impact of different pH conditions and temperature on viral infection.
Main Results:
- Bafilomycin A1 and concanamycin A effectively blocked influenza virus entry at low concentrations.
- Viral particle attachment to the cell surface was unaffected by bafilomycin A1.
- Nonspecific proton-ATPase inhibition also blocked entry, while plasma-proton ATPase inhibition had no effect.
- Both antibiotics inhibited infection under low-pH conditions, with reduced inhibition when cells were pre-bound at 4°C.
- Incubation at acidic pH alone potently blocked influenza virus infection.
Conclusions:
- Endosomal acidification, mediated by v-[H+]ATPase, is essential for efficient influenza virus entry into MDCK cells.
- A pH gradient across the endosomal membrane, rather than simply a low pH environment, is necessary for viral entry.
- These findings provide insights into the mechanism of influenza virus cell entry and potential therapeutic targets.