Here we developed a system to produce DENV pr peptide and reconstitute the pr-E interaction in vitro. At low pH pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion and trimerization. Addition of exogenous pr to mature DENV particles inhibited virus fusion and infection. Mutation of a key histidine residue in the pr-E interface, E H244, reduced pr's binding and inhibitory activity, and reduced DENV secondary infection and particle production. The defect in particle Figure 1 . Expression and purification of DENV2 pr and truncated E proteins. A) Linear diagrams of the DENV2 prM-E proteins and the truncated DENV2 pr and E proteins used in this work (not to scale). Domain and construct boundaries are marked, with numbering based on the individual proteins in the DENV2 New Guinea C (NGC) strain. The sequences appended to the diagrams contain the Strep (ST) affinity tag(s) used for protein purification (underlined), joined in the case of two Strep tags by a flexible linker region (STST). Pr was expressed in 293T cells and contains prM residues 1-86 plus N-terminal GS residues from the vector and the STST tag. The DI/II and E9 proteins were expressed in S2 cells and contain E residues 1-291 and 1-395, respectively, plus ST or STST tags. DIII was expressed in E. coli and contains E residues 289-430, comprising the linker, DIII, helix 1 and conserved sequence (LDIIIH1CS). The names in parentheses are the detailed nomenclature from [30] . B) 4 mg samples of purified pr peptide were incubated with DTT, Endo H, or PNGase F as indicated, analyzed by SDS-PAGE and stained with Coomassie blue. The positions of marker proteins are shown on the right with their molecular masses listed in kilodaltons. Asterisks indicate the positions of the added glycosidases. C) 4 mg samples of purified truncated E proteins were reduced with DTT as indicated, analyzed by SDS-PAGE and stained with Coomassie blue. Marker proteins are shown on the right with their molecular masses listed in kilodaltons. doi:10.1371/journal.ppat.1001157.g001