CHARACTERIZATION OF THE RUBELLA-VIRUS NONSTRUCTURAL PROTEASE DOMAIN AND ITS CLEAVAGE SITE

The region of the rubella virus nonstructural open reading frame that contains the papain-like cysteine protease domain and its cleavage sit e was expressed with a Sindbis virus vector, Cys-1151 has previously b een shown to be required for the activity of the protease (L. D. Marr, C,-Y. Wang, and T, K, Frey, Virology 198:586-592, 1994), Here we show that His-1272 is also necessary for protease activity, consistent wit h the active site of the enzyme being composed of a catalytic dyad con sisting of Cys-1151 and His-1272, By means of radiochemical amino acid sequencing, the site in the polyprotein cleaved by the nonstructural protease was found to follow Gly-1300 in the sequence Gly-1299-Gly-130 0-Gly-1301. Mutagenesis studies demonstrated that change of Gly-1300 t o alanine or valine abrogated cleavage. in contrast, Gly-1299 and Gly- 1301 could be changed to alanine with retention of cleavage, but a cha nge to valine abrogated cleavage, Coexpression of a construct that con tains a cleavage site mutation (to serve as a protease) together with a construct that contains a protease mutation (to serve as a substrate ) failed to reveal trans cleavage, Coexpression of wild-type construct s with protease-mutant constructs also failed to reveal trans cleavage , even after extended in vitro incubation following lysis. These resul ts indicate that the protease functions only in cis, at least under th e conditions tested.