FIG. 3.

Cell association of recombinant NLV particles and mapping mutations which ablate particle formation. To determine if VEE-Nor particles are preferentially secreted from infected cells or remain in cellular compartments, BHK cells (4 × 10⁷ cells) were infected with VRP-NV1 at a MOI of 2 for 1 h. At 36 h postinfection, supernatants were harvested, cell monolayers were frozen twice, and cell debris was removed by centrifugation. Virus particles were concentrated from the supernatants or cell lysates by centrifugation through 20% sucrose cushions for 3 h at 28,000 rpm in a Beckman SW28 rotor and analyzed by electron microscopy as described in the legend to Fig. 2. Intracellular (A) and extracellular (B) VEE-Nor and particle formation from VEE-NV3- (C) and VEE-NV4- (D) transfected BHK cells (8.0 × 10⁶). Arrows indicate NLV particles. Bars = 100 nm.