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Purification and characterization of the reovirus cell attachment protein sigma 1
Abstract:
It has previously been shown that of all the soluble reovirus-specified proteins present in the infected cell lysate, protein sigma 1 alone possesses the capacity to bind to host cells (P.W.K. Lee, E.C. Hayes, and W.K. Joklik, 1981, Virology 108, 156-163). We found that sigma 1 from urea-disrupted reovirus particles was also capable of such specific binding. Reovirions were therefore used as a source of functional sigma 1. Accordingly, a simple procedure has been developed to purify sigma 1 by subjecting urea-disrupted reovirions to DEAE ion-exchange chromatography. Protein sigma 1 thus isolated was electrophoretically homogeneous and the recovery was estimated to be 50 to 60% of the theoretical yield. The purified protein presumably maintained its native conformation since it was recognized by a panel of monoclonal anti-sigma 1 antibodies previously isolated, and was capable of specifically binding to host cell receptors, agglutinating human erythrocytes and inducing neutralization and hemagglutination-inhibition antibodies. Subsequent chemical crosslinking studies revealed the presence of oligomeric (mostly dimeric) sigma 1 forms in the preparation. The amino acid composition of the purified sigma 1 was found to closely match that inferred from the S1 gene sequence. However, attempts to determine its amino-terminal sequence have not been successful. The p/ of the purified protein was determined to be 6.8. Circular dichroic measurements of the purified sigma 1 indicated that 54 and 19% of its residues were arranged in alpha-helical and beta-sheet secondary structures, respectively.
Insights
Researchers purified reovirus protein sigma 1, finding it retains its host cell binding ability. This purified protein is crucial for understanding reovirus interactions and potential therapeutic targets.
Area of Science:
- Virology
- Molecular Biology
- Protein Chemistry
Background:
- Reovirus protein sigma 1 is known to bind host cells.
- Previous studies indicated sigma 1's unique cell-binding capacity among reovirus proteins.
Purpose of the Study:
- To develop a method for purifying functional reovirus protein sigma 1.
- To characterize the properties of purified sigma 1 and confirm its biological activity.
Main Methods:
- Purification of sigma 1 from urea-disrupted reovirions using DEAE ion-exchange chromatography.
- Electrophoretic analysis for purity assessment.
- Functional assays including host cell binding, hemagglutination, and antibody neutralization.
- Chemical crosslinking, amino acid composition analysis, and circular dichroism spectroscopy.
Main Results:
- A simple purification procedure yielded electrophoretically homogeneous sigma 1 with 50-60% recovery.
- Purified sigma 1 maintained native conformation, binding host cells, agglutinating erythrocytes, and inducing neutralizing antibodies.
- Crosslinking studies indicated the presence of oligomeric sigma 1, predominantly dimeric.
- Amino acid composition matched the S1 gene sequence, but N-terminal sequencing was unsuccessful.
Conclusions:
- Functional reovirus protein sigma 1 can be efficiently purified using ion-exchange chromatography.
- The purified sigma 1 retains key biological activities, including host cell receptor binding and immunogenicity.
- Structural analysis revealed alpha-helical and beta-sheet content, providing insights into sigma 1's conformation.