FIG. 2.

CVB3 infection (MOI of 50) and expression of the 3A protein inhibit A1PI secretion. BGM cells were transfected with A1PI expression constructs. Twenty hours after transfection, the cells were starved of methionine for 30 min, labeled with [³⁵S]methionine for 30 min, and then chased for 2 h. Cell (C) and medium (M) fractions were collected and analyzed for the amount of labeled reporter protein by immunoprecipitation with an anti-EGFP antiserum, SDS-PAGE, and phosphorimaging. (A) Cells transfected with a plasmid containing A1PI behind the PV noncoding region were infected and subjected to pulse-chase analysis at 2, 4, and 6 h p.i. The differences in migration between A1PI in the medium and cell fractions were due to differences in the glycosylation state (endoglycosidase F treatment resulted in faster migration of A1PI in both the medium and cell fractions [data not shown]). (B) Average secretion (means ± standard errors of the means [SEM]) of three independent experiments. A1PI secretion was calculated as the percentage of A1PI secretion in uninfected control cells, which was normalized to 100%. (C to E) BGM cells were transfected with constructs expressing either EGFP (−3A) or EGFP-3A (+3A) from an SV40 promoter and the A1PI-EYFP protein from a cytomegalovirus promoter. (C) Western blot analysis of EGFP and EGFP-3A expression. (D) Analysis of A1PI secretion. (E) Average secretion (means ± SEM) of three independent experiments. A1PI secretion was calculated as the percentage of A1PI secretion in EGFP-expressing control cells, which was normalized to 100%.