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Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein
M R Shapouri1, M Arella, A Silim
1Section de virologie, Faculté de Médecine vétérínaire, Université de Montréal, Québec, Canada.
Abstract:
It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.
Insights
Avian reovirus sigma 3 protein forms multimers and binds to cells, similar to mammalian reovirus sigma 1 protein. This binding is specific and crucial for initiating viral infection.
Area of Science:
- Virology
- Molecular Biology
- Protein Structure
Background:
- Avian reovirus sigma 3 protein is hypothesized to be analogous to mammalian reovirus sigma 1 trimer, a key attachment protein.
- Understanding the multimeric state and cell-binding properties of sigma 3 protein is essential for elucidating its role in viral entry.
Purpose of the Study:
- To investigate the multimeric nature of avian reovirus sigma 3 protein.
- To determine the cell-binding ability of sigma 3 protein.
- To assess the role of sigma 3 protein in the initiation of viral infection.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-dissociating conditions to assess protein multimerization.
- Expression of sigma 3 fusion protein (sigma 3-MBP) in Escherichia coli and COS-7 cells.
- Cell attachment assays using Vero cells.
- Inhibition assays using monoclonal antibodies and competing virus particles.
- Plaque-forming unit (p.f.u.) reduction assays.
Main Results:
- Sigma 3 protein exists as a multimer in its undisrupted form.
- Virion-associated and cell-expressed sigma 3 protein showed different behaviors in SDS-PAGE, suggesting regulation by virus-associated factors.
- Escherichia coli-expressed sigma 3-MBP, in its multimeric form, specifically attached to Vero cells in a receptor-mediated manner.
- Sigma 3-MBP binding was inhibited by anti-sigma 3 monoclonal antibodies and competing avian reovirus particles.
- Sigma 3-MBP reduced virus p.f.u. in cell cultures, confirming its role in infection initiation.
Conclusions:
- Avian reovirus sigma 3 protein is a multimer capable of specific, receptor-mediated cell attachment.
- Virus-associated factors likely regulate the multimerization of sigma 3 protein.
- Sigma 3 protein plays a critical role in the initiation of avian reovirus infection.