We first tested the effect of the H244A mutation on pr-E binding. In agreement with our earlier results, WT DI/II protein was efficiently pulled-down by pr-sepharose (Fig. 5B ). Pull-down was low pH-dependent and blocked by mAb 4G2 against the E fusion loop at the DII tip. In contrast, almost no H244A DI/II protein was pulled-down by pr-sepharose at either low pH or neural pH (Fig. 5B ). SPR analysis of WT DI/II protein showed most efficient binding at pH 6.0, and binding was blocked by preincubating the DI/II protein with mAb 4G2 (molar ratio 1:1) before dilution into SPR buffer (Fig. 5C, upper panel) . Equivalent concentrations of H244A DI/II protein showed greatly reduced binding to pr compared to that of WT protein (Fig. 5C , lower panel). Although H244A binding was decreased, the residual binding was still blocked by mAb 4G2 and had an acidic pH optimum. This suggests that binding also involves other residues in the pr-E interface, such as the complementary residues identified in the structural studies and shown in Fig. 5A .