As a first test of in vitro pr-E binding, we coupled pr to sepharose beads and tested its ability to pull-down truncated E protein containing only domains I and II. This form of E protein is monomeric and the tip of DII is thus accessible even at neutral pH. Previous studies showed that this and other DENV DI/II proteins are active in membrane insertion and trimerization at both neutral and low pH [30] . We observed efficient pull-down of DI/II protein by pr-sepharose ( Fig. 2A) , but in spite of the accessibility of the pr binding site on DI/II at neutral pH, pulldown was low pH-dependent. The pull-down of DI/II protein by pr was specific, as it was blocked by inclusion of mAb 4G2 against the E fusion loop at the DII tip, and did not occur with BSAsepharose beads. These data suggested that the recombinant pr peptide could bind to the tip of DI/II in a low pH-dependent reaction.