The Post-Translational Processing of Sindbis Virus Glycoproteins

Citation

Mayne, Jeffery Terrell (1984) The Post-Translational Processing of Sindbis Virus Glycoproteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jvbk-es85. https://resolver.caltech.edu/CaltechTHESIS:11282018-124040200

Abstract

A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus infected chicken cells and shown to be produced from the cleavage of PE2 to produce E2. The N-terminal sequence of E3 is identical to that of PE2. The first 19 amino acids are hydrophobic and presumably serve as the signal sequence for PE2. This sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at position 14. Labeling studies imply that the PE2 → E2 + E3 cleavage is not closely coupled to budding. E3 is cleaved and released into the culture fluid under conditions where no virions bud, and the kinetics of appearance of E3 in the culture fluid and E2 in virions are dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane glycoproteins.

Hybridomas were selected by the fusion of NSI/1 myeloma cells with spleen cells from mice inoculated with Sindbis specific antigens. Ten stable hybridomas were obtained, seven producing E1-specific antibodies and three producing capsid-specific antibodies. The seven E1 specific antibodies were divided into two classes, which reacted with different E1 antigenic domains. The two classes of antibodies differed in several tested properties. Two E1 clones inhibited viral infectivity, and one of these precipitated E2 along with E1 in Triton-treated preparations. These properties are discussed with regard to the known relationships between the viral structural proteins.

The tryptic glycopeptides of E1 and E2 grown in BHK or chick cells were purified and analyzed by N-terminal sequencing, pronase digestions and labeling with various radioactive sugars. We found that the glycosylation patterns for the two proteins were essentially identical in the two hosts. E2 contains exclusively complex chains attached to Asn₁₉₆ and simple chains attached to Asn₃₉₈. In E1, the Asn₁₃₅ glycosylation site contained only complex chains, but the Asn₂₄₅ site contained a mixture of simple and complex chains. A prediction as to the relative importance of the different glycosylation sites to protein function is offered.

Thesis Files