Efficient in vitro translation and processing of the rubella virus structural proteins in the presence of microsomes.
In the structural protein open reading frame (SP-ORF) of rubella virus (RUB), the sequences for the three virion proteins occur in the order NH2-C-E2-E1-COOH with hydrophobic, consensus signal sequences preceding the amino termini for each of the two membrane proteins (T. K. Frey and L. D. Marr, 1988 Gene 62, 85-100). In vitro translation in the presence of microsomes of RNA transcripts from a plasmid containing the SP-ORF resulted in production and accurate processing of the three structural proteins. Since in the absence of microsomes the 110-kDa precursor of these proteins is produced, this finding indicated that the cleavage events in processing of the precursor were mediated by signalase. To study the C-E2 processing event, a DNA construct was made which contained the sequences for E2 beginning at the NH2 terminus of the hydrophobic consensus signal and extending through to the NH2 terminus of E1. In vitro translation of transcripts from this construct in the presence of microsomes resulted in accurate processing of E2 confirming that the hydrophobic sequence was a signal sequence and demonstrating it could function externally as well as internally within the 110-kDa precursor. To determine if the E2 signal was maintained on C after cleavage of the precursor by signalase, the SP-ORF plasmid was mutagenized to place translation termination codons at either the NH2 or COOH side of the E2 signal sequence such that C protein lacking or containing the E2 signal would be produced. As expected, the C-minus-signal protein migrated more rapidly in polyacrylamide gels than did the C-plus-signal protein. C translated from the SP-ORF construct as well as authentic C from infected cells comigrated with the C-plus-signal protein, indicating that the E2 signal was not removed. In a corollary study, it was found that RUB C protein was phosphorylated in vivo, although the percentage of the protein phosphorylated was not determined.
Marr
L D
LD
Department of Biology, Georgia State University, Atlanta 30302-4010.
Sanchez
A
A
Frey
T K
TK
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008861:Microsomes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012412:Rubella virus / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D015678:Viral Structural Proteins