FIG. 5.

Folding stability of the envelope proteins of FrCas^NC and F43 were assessed by the extent of binding of the ER chaperone BiP. NIH 3T3 cells were infected with F43 or FrCas^NC, and the cells were lysed 48 h later. Lysates were immunoprecipitated (IP) with anti-SU or anti-BiP (anti-KDEL). Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted (IB) with either anti-SU or anti-BiP antibodies. The envelope protein is synthesized in the ER as a polyprotein pr85^env (asterisks), which after transit to the Golgi compartment is cleaved by a cellular protease into SU (dot) and TM (not shown) components (see top Fig. 2). The smaller molecular sizes of the pr85^env and SU proteins of FrCas^NC compared to those of F43 are due to two deletions of 4 and 7 amino acid residues in the PRR (see Fig. 2) of CasBrE. The envelope protein of FrCas^NC but not F43 is retained in the ER, which accounts for the difference in the ratio of pr85^env and SU proteins observed in F43 and FrCas^NC-infected cells (IP-SU/IB-SU). The increased binding of BiP (arrowheads) to the pr85^env protein of FrCas^NC relative to that of F43 (IP-SU/IB-BiP and IP-BiP/IB-SU) is consistent with the folding instability of the FrCas^NC envelope protein reported previously (9).