Biosynthetic radiolabeling of virus glycoproteins for immunoprecipitation and electrophoretic analysis.

Abstract

AbstractThere are many different approaches to a biochemical analysis of virus glycoproteins. However, because Epstein-Barr virus (EBV) does not replicate to high titer in any cell line, several of these are compromised by the relatively small amounts of glycoprotein that are made. For example, unless expressed to high levels under the control of a heterologous promotor, most EBV glycoproteins are not made in sufficient quantity to be detected by Western blotting. Virus expression is not induced at high enough levels in the maximal amount of cell protein that can be reasonably loaded on a standard one dimensional gel (equivalent to approx two million cells). Likewise, the inability to induce virus replication in the majority of latently infected cell lines in a highly synchronous manner makes it difficult to incorporate sufficient amounts of radiolabel into glycoproteins in a short period of time in order to perform pulse chase analyses. Long-term biosynthetic radiolabeling of glycoproteins does, however, allow for analysis by immunoprecipitation and electrophoresis and in combination with inhibitors of glycosylation such as tunicamycin or glycosidases such as endoglycosidase N and endoglycosidase F can provide a considerable amount of useful information about the processing and biochemical characteristics of a glycoprotein species. Glycoproteins have been analyzed in each of the cell lines that can be induced to produce virus. However, the Akata cell line (1), which can be induced in a relatively synchronous manner (2) by treatment with anti-human immunoglobulin to support virus replication in more than 30% of cells (3), provides the best results. KeywordsLytic CycleComplete RPMI MediumHeterologous PromotorTrypan Blue SolutionInfected Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.