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. 2006 Sep 11;184(1):23–32. doi: 10.1016/0042-6822(91)90818-V

Biochemical and biophysical characterization of the reovirus cell attachment protein σ1: Evidence that it is a homotrimer

James E Strong *, Gustavo Leone *, Roy Duncan *, Rajendra K Sharma †, Patrick WK Lee *,1
PMCID: PMC7130766  PMID: 1871968

Abstract

The oligomerization state of the reovirus cell attachment protein σ1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus Sl mRNA transcripts synthesizedin vitro were cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of σl. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified σ1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of σ1 and allowed us to determine the molecular weight of the native protein to be ∼132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the σ1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein σ1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that σ1 might be a higher order oligomer.

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