Mutational analysis of the arginine residues in the E2-E1 junction region on the proteolytic processing of the polyprotein precursor of rubella virus.
Endoproteolytic cleavage of precursors is a key step in biosynthesis of functional proteins. The structural proteins of rubella virus are initially translated as a precursor polyprotein in the order NH2-C-E2-E1-COOH and are cleaved by host signal peptidase to yield three structural proteins. Between regions corresponding to E2 and E1 in the precursor is a region of seven amino acid residues (R-R-A-C-R-R-R) that contains a motif for stop-transfer or a possible target for trypsin-like protease cleavage. Using site-directed mutagenesis, these arginine residues, as well as the signal peptide cleavage site at the N-terminus of E1, have been mutated individually or in combination. Results from in vitro transcription/translation analysis indicated that the mutated E2E1 precursor polyproteins were translocated into the microsome and glycosylated. Expression of mutated precursor polyproteins in COS cells revealed that the cleavage of E2E1 polyprotein precursor was impaired when the signal peptide cleavage site alone or both arginine clusters were altered, whereas partial cleavage was observed in the mutants in which either one of the two arginine clusters was modified. Our data suggest that although the arginine clusters do not function as a basic protease cleavage site, they contribute to maintain the proper configuration of that region for access of cellular signal peptidase.
Qiu
Z
Z
Department of Pathology, Faculty of Medicine, University of British Columbia Research Centre, Vancouver, Canada.
McDonald
H L
HL
Chen
J
J
Hobman
T C
TC
Gillam
S
S
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D012412:Rubella virus / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
D011498:Protein Precursors; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; C061826:E1 envelope protein, Rubella virus; C061827:E2 envelope protein, Rubella virus; D001120:Arginine