FIG. 4.

Characterization of vA14(N83Q). (A) The A14 protein encoded by vA14(N83Q) is not glycosylated in vivo. Cells were infected with vindA17 (without IPTG) (lane 1), wt virus (lane 2), or vA14(N83Q) (lane 3) and were metabolically labeled with [³⁵S]methionine from 6 to 9 hpi. Cell lysates were subjected to immunoprecipitation with anti-A14 serum; immunoprecipitates were resolved by SDS-PAGE and were visualized by fluorography. As above, the symbols indicate unmodified A14 (◂), glycosylated A14 (shaded circle), and coprecipitated A17 protein (▴). (B) Determination of CAV and ECV yields. Cells were infected at an MOI of 2 with wt virus in the absence or presence of BFA or with two isolates of vA14(N83Q). At 24 hpi cells and culture media were harvested and viral yield was determined by plaque assay. CAV is largely IMV but also scores low levels of IEV and cell-associated enveloped virus (see introduction). The black horizontal line, set at the titer of virus found in the supernatant fluid of cultures infected in the presence of BFA, represents the background level of IMV that had leaked into the supernatant fluid. Titers above this threshold are interpreted as representing bona fide EEV. No significant differences are seen in the levels of CAV or ECV produced by vA14(N83Q) versus those produced by wt virus.