FIG 3.

NSP2 detected in mock- and rotavirus-infected cell lysates by Western blotting and modeling the disulfide bond in NSP2. (A and B) Sample lysates prepared with β-mercaptoethanol (βME) and urea (+) (A) or prepared without βME or urea (−) (B) were boiled for 3 min and analyzed by gradient SDS-PAGE. Proteins in the resulting gels were transferred to nitrocellulose and probed for NSP2 with either MAb dNSP2_NT or MAb vNSP2_CT hybridoma supernatant and detected with goat anti-mouse secondary antibodies labeled with alkaline phosphatase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection was used as a loading control. The molecular masses (in kilodaltons) are shown on the right of each panel. (C) Licorice representation of NSP2 monomer is shown with the N- and C-terminal domains colored in green and blue, respectively. Four cysteine residues in NSP2 are shown as spheres and indicated by black arrows. (D) The side chains of a pair of Cys residues (Cys8 and Cys85) in NSP2 structure (PDB accession no. 1L9V) are close enough to engage in disulfide bond formation with small changes in the side chain orientations. The side chains of Cys are represented with balls and sticks, with their carbon and sulfur atoms colored red and yellow, respectively. (E) Modeling of a possible disulfide bond between Cys8 and Cys85 by rotating their side chains. The side chains are colored by the same scheme as described above for panel D.