Figure 1

Mutation of S40 to Phe, which does not alter the amino acid sequence in the overlapping pol reading frame, nevertheless inhibits CA maturation, alters budding, and reduces viral infectivity. Panels A1-A3, Western blot analysis of pNL4-3ΔEnv WT, S40A and S40F constructs which encode active protease. VLPs and cell lysates from COS-1 cells transfected with pNL4-3ΔEnv-WT (lane 1), pNL4-3ΔEnv-S40A (lane 2) or pNL4-3ΔEnv-S40F (lane 3) were analyzed by Western blotting. The values in the figure indicate the ratio of p25/p24 normalized to wild type. The relative VLP efficiencies [VLP/(VLP + Gag from cell lysate)] were determined as described in Methods. Panel B, Electron microscopy of particles associated with cells transfected with pNL4-3ΔEnv-WT (panel B1), or S40F (panel B2). Immature particles (open arrows), particles with aberrant cores (gray arrows), and mature particles with conical cones (solid arrows) were released from pNL4-3ΔEnv-transfected cells. Panel C, Quantitative analysis. 25 WT or 100 S40F VLPs (n =2) were counted and the frequency of each type of particle was determined. Panel D, Viral particle infectivity, determined by MAGI assay, done in triplicate.