Figure 3

Analysis of the I5 protein found within mature virions. (A) I5 is encapsidated within mature virions. Increasing concentrations of purified I5V5 virions (0.5, 0.8, 2 μg) were subjected to immunoblot analysis and probed with the anti-V5 antibody. The 9 kDa I5V5 protein was readily visualized; the molecular masses (in kDa) of protein standards are shown at the right. (B). I5 is found within the virion membrane. wt and I5V5 virions purified by sedimentation on 25–40% sucrose gradients were treated with NP40 or NP40 and DTT (5). The soluble (S) and particulate (P) fractions, representing the membrane and core components, respectively, were resolved by sedimentation and analyzed by immunoblot analysis with anti-V5 and antibodies against known membrane (A17) and core (F18) proteins. (C) The V5 epitope is accessible on the surface of intact I5V5 virions. Purified vI5V5 virions (or a control virus encoding a wt I5 protein lacking the V5 epitope) were applied to grids and probed with either a control antibody (anti-A17) or the anti-V5 antibody and a secondary antibody conjugated to 10 nm gold particles. (D). Proteolytic treatment of vI5V5 virions. I5V5 virions (6 μg) were subjected to treatment with chymotrypsin (Chymo) or trypsin (Tryp) for 30 min or with proteinase K (ProtK) for 10 or 30 min. After sedimentation at 14,000 × g, 5 min, the soluble (S) and pellet (P) fractions were resolved and analyzed by immunoblot analysis using anti-D8 (top panel) or anti-V5 (lower panel) antibodies. The molecular masses (in kDa) of protein standards are shown at the right.