Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins.

Abstract

Sindbis virus 26S RNA was translated in a cell-free protein-synthesizing system from rabbit reticulocytes. When the system was supplemented with EDTA-stripped dog pancreas microsomal membranes, the following results were obtained: Complete translation of 26S RNA, resulting in the production, by endoproteolytic cleavage, of 3 polypeptides that are apparently identical to those forms of C [capsid protein], PE2 [precursor protein] and E1 [glycoprotein] that are synthesized in vivo by infected host cells during a 3 min pulse with [35S]methionine. Corrected topological deposition of the 3 viral polypeptides-in vitro-synthesized PE2 and E1 forms are inserted into dog pancreas microsomal membranes in an orientation which, by the criterion of their limited (or total) inaccessibility to proteolytic probes, is indistinguishable from that of their counterparts in the rough endoplasmic reticulum of infected host cells; in vitro-synthesized C is not inserted into membranes and therefore is accessible to proteolytic enzymes, like its in vivo-synthesized counterpart. Core glycosylation of in vitro-synthesized PE2 and E1 forms, as indicated by binding to concanavalin A Sepharose and subsequent elution by .alpha.-methylmannoside.