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Published April 1984 | public
Journal Article

Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3


A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus-infected primary chick embryo fibroblasts. Tryptic peptide mapping and pulse-chase studies verified that this protein was produced as a by-product of the cleavage of the precursor protein PE2 to produce the envelope glycoprotein E2. A 2600-fold purification was achieved via a procedure which used differential ethanol precipitation, gel filtration, ion-exchange chromatography, and affinity chromatography on a lentil lectin column. Amino acid composition analysis, N-terminal microsequencing, and labeling studies yielded information about the fine structure of E3 and its relationship to E2 and virion maturation. The N-terminal sequence of E3 is identical to that of PE2, including the result that 90% of the molecules appear to be blocked. The first 19 amino acids are uncharged and presumably serve as the signal sequence for the insertion of PE2 into the membrane of the endoplasmic reticulum, but this sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at the asparagine at position 14. The two residues at the C-terminus of E3, Lys-Arg, are removed during or shortly after cleavage from PE2. Labeling studies imply that, although the PE2 -> E2 + E3 cleavage is necessary for virion budding, these two events are not closely coupled. E3 is cleaved and released into the culture fluid under conditions where virions do not bud, and the kinetics of the appearance of ES in the culture fluid and E2 in virions are quite dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane and secretory glycoproteins.

Additional Information

© 1984 Academic Press, Inc. Received October 28, 1983; accepted January 20, 1984. We would like to thank E. Lenches for preparing monolayers of chicken embryo fibroblasts and large quantities of supernatants from polyethylene glycol precipitations of Sindbis virus; J. R. Bell for his advice and expertise; and L. Hood for his generosity in lending us his equipment. This work was supported by Public Health Service Grants GM 06965 and AI 10793 from the National Institutes of Health and Grant PCM 80 22830 from the National Science Foundation. J. T. Mayne was supported by Training Grant GM 00086 from the National Institutes of Health.

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