Location of Intrachain Disulfide Bonds in the VP5 * and VP8* Trypsin Cleavage Fragments of the Rhesus Rotavirus Spike

and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simian rotavirus SAll in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5 * and VP8 * possessed intrachain disulfide bonds. Given that the VP8* fragments ofRRV and SAll contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys- Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8 * of RRV were linked by a disulfide bridge. VP5 * expressed in vitro was able to form a disulfide bond analogous to that in the VP5 * fragment of trypsin-treated RRV. Analysis of a Cys-774->Ser mutant ofVP5 * showed that, while it was able to form a disulfide bond, a Cys-318- Ser mutant of VP5 * was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5 * region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain ofVP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and anima