FIG. 3.

Entry pathways. (A) Inhibition of the major cellular entry pathways during PPV infection. Optimal concentrations of inhibitors of clathrin endocytosis (chlorpromazine and K⁺ depletion) (light shaded bars), caveolae (nystatin and methyl-β-cyclodextrin) (filled bars), and macropinocytosis (amiloride and cytochalasin D) (dark shaded bars) were used. The percentage of cells infected was determined at 20 h by indirect IF experiments as described for Fig. 2. The combination of clathrin and macropinocytosis inhibitors (striped bars) failed to completely abolish infection (**, P < 0.003 by the t test). (B) Inhibition of genome replication measured by qPCR with specific PPV primers after treatment with inhibitors as for panel A (*, P < 0.03 by the t test). (C) Binding assay. Inhibitors were present only for the first 2 h with PPV at 4°C and were removed at the time of washing to eliminate unbound virus. The infection was continued in normal cell culture medium for 20 h. (D) Pulse inhibition. Clathrin endocytosis and macropinocytosis inhibitors either were added prior to infection and were present only for the first 2 h of infection, after which cells were washed; were added 2 h p.i.; or were added prior to infection and left during the whole infection (20 h). (E) Inhibition of distinct particle types. Shown are results for crude preparations (infected cell culture supernatants), isolated particles and aggregates (both separated by low-speed centrifugation of the crude preparation), and purified particles. To determine the inhibition of the clathrin and macropinocytosis pathways for each particle type, infections were carried out with the same amount of PPV, as determined by qPCR (***, P < 0.0003 by the t test).