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Intracellular processing of measles virus fusion protein
T A Sato1, T Kohama, A Sugiura
1Department of Measles Virus, National Institute of Health, Tokyo, Japan.
Abstract:
Intracellular processing of measles virus fusion (F) protein was studied by radiolabeling and immunoprecipitation with a monoclonal antibody against F protein. The cleavage of F protein into F1 and F2 subunits was complete after 5 hours of chase during which the growth of oligosaccharide chains on the F2 domain of F protein continued. The addition of terminal sialic acid conferred a strong negative charge on the F2 subunit. F protein expressed on the cell surface was removed by a fungal semi-alkaline protease, providing a method to follow the kinetics of its transport to the cell surface. The transport of the F protein was faster than that of the hemagglutinin (HA) protein. Uncleaved F protein, as well as cleaved subunits became digestible by the protease, indicating that a portion of the F protein reaches the cell surface uncleaved. The treatment of measles virus-infected cells with tunicamycin resulted in the synthesis of unglycosylated HA (65 kilodaltons, Kd) and F (48 Kd) proteins. Unglycosylated F protein was not cleaved into smaller subunits, nor was it transported to the cell surface. Unglycosylated HA protein likewise failed to reach the cell surface.
Insights
Measles virus fusion (F) protein undergoes cleavage and glycosylation during intracellular processing. Proper glycosylation is essential for F protein transport to the cell surface.
Area of Science:
- Virology
- Cell Biology
- Protein Biochemistry
Background:
- Measles virus fusion (F) protein is critical for viral entry.
- Understanding F protein intracellular processing is key to developing antiviral strategies.
Purpose of the Study:
- To investigate the intracellular processing, cleavage, and cell surface transport of measles virus F protein.
- To determine the role of glycosylation in F protein maturation and transport.
Main Methods:
- Radiolabeling and immunoprecipitation using a monoclonal antibody against F protein.
- Protease digestion assay to assess cell surface expression and cleavage.
- Tunicamycin treatment to study the effects of inhibiting glycosylation.
Main Results:
- F protein cleavage into F1 and F2 subunits occurred within 5 hours, with continued oligosaccharide growth on F2.
- Terminal sialic acid addition imparted a negative charge to the F2 subunit.
- F protein transport to the cell surface was faster than hemagglutinin (HA) protein transport.
- Unglycosylated F protein, synthesized after tunicamycin treatment, was not cleaved or transported to the cell surface.
- Both uncleaved and cleaved F proteins were susceptible to protease digestion at the cell surface.
Conclusions:
- Measles virus F protein undergoes proteolytic cleavage and glycosylation during intracellular maturation.
- Glycosylation is indispensable for the proper folding, cleavage, and cell surface transport of F protein.
- A portion of F protein reaches the cell surface in an uncleaved state.