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 site was expressed with a Sindbis virus vector. Cys-1151 has previously been 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 with the active site of the enzyme being composed of a catalytic dyad consisting 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-1300-Gly-1301. Mutagenesis studies demonstrated that change of Gly-1300 to alanine or valine abrogated cleavage. In contrast, Gly-1299 and Gly-1301 could be changed to alanine with retention of cleavage, but a change to valine abrogated cleavage. Coexpression of a construct that contains 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 constructs with protease-mutant constructs also failed to reveal trans cleavage, even after extended in vitro incubation following lysis. These results indicate that the protease functions only in cis, at least under the conditions tested.
Chen
J P
JP
Department of Biology, Georgia State University, Atlanta, Georgia, 30303, USA.
Strauss
J H
JH
Strauss
E G
EG
Frey
T K
TK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D012412:Rubella virus / Q000201:enzymology*; D012845:Sindbis Virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D006639:Histidine; D010450:Endopeptidases