Previous studies showed that retention of endogenous pr peptide on the furin-processed DENV particle inhibits virus interaction with liposomes at low pH [35] . Structural considerations suggested that this inhibition occurs primarily by blocking low pH-triggered dissociation of the E dimer, a required first step in the fusion reaction. To test this mechanism, we evaluated the effect of pr on the membrane interactions of dimeric and monomeric forms of E protein. The E9 dimer was preincubated with pr peptide or an unrelated protein with the same affinity tag for 5 min at pH 8.0, and then treated at pH 5.75 in the presence of target liposomes. Membrane-associated proteins were separated by liposome floatation on sucrose gradients. There was no liposome cofloatation when E9 protein was incubated with liposomes at neutral pH (Fig. 3A) . About 70% of the total E9 floated with liposomes in the top part of the sucrose gradient after treatment at pH 5.75 in the presence (Fig. 3A , top panel) or absence (data not shown) of a control protein. In contrast, when E9 was preincubated with pr peptide (pr:E9 molar ratio 12:1) and treated with low pH, only ,2% of E9-ST floated with the liposomes (Fig. 3A , middle panel). Inhibition by pr was not observed when it was added after E9 was treated at low pH in the presence of liposomes for 30 min (Fig. 3A , bottom panel), and thus pr needed to be present during the membrane insertion step. Inhibition was concentrationdependent, with 22% E9 co-floatation at a pr:E9 molar ratio of 3:1, 8% at 6:1, and 0.4% for 24:1 (data not shown; see also We then tested the effect of pr on the DENV E DI/II protein. This protein is monomeric and its stable membrane interaction requires DIII to ''clamp'' the core trimer [30] . As shown in Fig. 3B , ,25% of DI/II co-floated with liposomes at low pH in the present of DIII, while no co-floatation was detected when BSA was Figure 3 . Pr peptide inhibits E protein-membrane interaction. A) E9-liposome co-floatation assay. E9 protein was mixed with pr peptide or an ST-tagged control protein (Seap) at a final concentration of 50 mg E9 protein and 200 mg pr/Seap protein/ml (molar ratio 12 pr/1E). Liposomes were added at a final concentration of 1 mM, and the samples were incubated at the indicated pH for a total of 60 min at 28uC. Where indicated, E9 protein plus liposomes were incubated for 30 min, pr peptide added to a final concentration of 200 mg/ml, and the incubation continued for an additional 30 min. The liposome-bound proteins were then separated by floatation on sucrose gradients at the indicated pH. Aliquots of the top, middle and bottom of the gradients were analyzed by SDS-PAGE and western blotting for E protein. B) DI/II-liposome co-floatation assay. 40 mg/ml DI/II plus DIII or BSA (200 mg protein/ ml) were incubated with liposomes plus 200 mg pr peptide/ml as indicated and assayed for liposome co-floatation as in panel 3A. C) SFV DI/II-liposome co-floatation assay. SFV DI/II protein (40 mg/ml) was mixed with BSA or pr peptide (160 mg/ml). Liposomes were added at a final concentration of 1 mM, and the samples were incubated at the indicated pH for a total of 30 min at 28uC. Liposome co-flotation was assayed as in panel 3A. D-E) Loss of pr inhibition of E protein in the pH range of the late endocytic pathway. D) pH dependence of pr inhibition. E9 protein was mixed with liposomes in the presence or absence of pr peptide (molar ratio ,12 pr/1E), treated at the indicated pH as in The structurally related alphavirus protein SFV E1 DI/II is monomeric and efficiently interacts with membranes at low pH (80% cofloatation, Fig. 3C , middle panel). No inhibition occurred when pr peptide was added prior to liposome addition (Fig. 3C , bottom panel), in keeping with the lack of pr-SFV DI/II binding in the SPR experiments discussed above. Thus, pr peptide specifically inhibits target membrane interaction of both monomeric and dimeric forms of the DENV E protein.