Processing of rotavirus glycoprotein VP7: implications for the retention of the protein in the endoplasmic reticulum.
Rotaviruses are icosahedral particles that assemble in the lumen of the endoplasmic reticulum (ER). The viral glycoprotein, VP7, is also directed into this compartment and is retained for assembly onto the surface of viral cores. VP7 is therefore a resident ER glycoprotein with a luminal orientation. The VP7 gene possesses two potential in-frame initiation codons, each preceding a hydrophobic domain. Mature VP7 is derived from a precursor by cleavage but the site of cleavage has not been determined because viral VP7 has a blocked amino terminus. Using site-directed mutagenesis of the gene and in vitro transcription and translation systems, we have investigated the synthesis and processing of the primary products synthesized from each initiation codon. Proteins translated from either codon were processed in vitro to yield products indistinguishable in size. The primary translation products therefore appeared to be cleaved at the same site. The site was located empirically between Ala50 and Gln51 and mutation of the gene to convert Ala50----Val prevented processing. Amino-terminal sequence analyses of proteins synthesized in vitro, and characterization of an amino-terminal fragment of VP7 purified from virus unequivocally established Gln51 as the amino-terminal residue. Pyroglutamic acid was tentatively identified as the blocking group. Processing of VP7 therefore removes both amino-terminal hydrophobic domains from the protein. Some other mechanism not requiring the presence of these hydrophobic sequences must account for the retention of this novel glycoprotein in the ER.
Stirzaker
S C
SC
Division of Molecular Biology, Commonwealth Scientific and Industrial Research Organisation (CSIRO), North Ryde, New South Wales, Australia.
Whitfeld
P L
PL
Christie
D L
DL
Bellamy
A R
AR
Both
G W
GW
D002451:Cell Compartmentation; D002460:Cell Line; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012401:Rotavirus / Q000378:metabolism*
D006023:Glycoproteins