Fig 2.

Ectopically expressed NS1 RQ10YK showed cellular targeting similar to that of NS1 RQ10NK. (A) BHK21-15 cells were infected with recombinant SINV (MOI of 10) ectopically expressing NS1 RQ (wild type), NS1 RQ10NK, or NS1 RQ10YK. At 14 h after infection, surface NS1 and total NS1 were measured by flow cytometry (middle panels) after sequentially incubating intact and permeabilized cells with different anti-NS1 MAbs (see Materials and Methods). Isotype controls also were performed (top panels). As an infectivity control, total SINV E1 expression level was measured using a polyclonal anti-E1 antibody (bottom panels). Contour plots are shown and are representative of three independent experiments. (B) Biosynthesis of wild-type and mutant NS1. BHK21-15 cells were infected at an MOI of 5 with SINV that ectopically expressed NS1 RQ (wild type), NS1 RQ10NK, or NS1 RQ10YK. At 15 h after infection, cells were placed in cysteine-methionine-free medium for 20 min, pulsed with [³⁵S]cysteine-methionine, and then chased for 1, 2, or 3 h. Cell supernatants or lysates were harvested, immunoprecipitated with 4NS1 MAb and protein A-Sepharose beads, and electrophoresed. The data are the representative results of two independent experiments.