Alanine mutagenesis experiments support the model for HeV-G mediated HeV-F activation. However, it should be pointed out that our structure based observations were derivedusing a soluble form of the HeV-G glycoprotein and would not take into account potential long-range effects on the structure of G or its native tetrameric configuration because of the absence of the stalk, transmembrane domain and cytoplasmic tail. Finally, our studies document that targeted mutations, such as I588A, can be designed in the henipavirus attachment proteins, which appear to uncouple host cell/ephrin attachment and viral fusion initiation, providing important tools for additional functional and structural analysis of the precise molecular steps underlying the receptor triggering mechanism of henipavirus entry, and how such mutations in the G attachment glycoprotein accomplish this block. The structure-based models discussed above also shed light on the dynamic process of the virus-receptor attachment stage, as well as on the mechanism of the G glycoprotein initiated fusionpromotion step that facilitates F activation. The structures provide a clear basis to design further experiments for evaluation of models of henipavirus attachment and entry into host cells and to examine certain key steps in the paramyxovirus entry process in general. Further, the series of structure-based point mutations made in the HeV-G glycoprotein underscore the importance of the individual residues forming the ephrin binding interface, and indicate that both the crystallographically-observed hydrophobic and electrostatic GSK-J4 interactions are crucial not only for cell attachment, but also for the subsequent membrane fusion and viral entry events. In addition, our structure-based mutagenesis experiments reveal three important and unique features of the HeV entry process: First, that single substitutions of interface GW788388 resides affect more strongly the HeV-G binding and entry to ephrin-B3-expressing cells as compared to ephrin-B2-expressing cells, suggesting that the HeV-G/ephrin-B2 attachment is more robust and resistant to minor structural perturbations; Second, that that the stability of the HeV-G/ephrin association does not strongly correlate with the efficiency of viral entry, suggesting that, particularly for ephrin-B2expressing cells, viral attachment is not the rate limiting step in the viral entry process; And third, that it is possible to alter the HeVG/ephrin interface in a way that does not affect the overall stability and/or affinity of the association, but that does affect the efficiency of viral entry, in the context of henipavirus pseudovirions, supporting a model where subtle structural changes at the HeV-G/ephrin interface are relayed at a distance to trigger largeFigure 9. Interaction of HeV-G mutants with HeV-F. (A) HeV-G mutants were co expressed with S tagged HeV-F in HeLa-USU cells. Lysates were immunoprecipitated (IP) with rabbit polyclonal G-specific antiserum to evaluate total G expression (top panel) or S agarose to evaluate total F expression (bottom panel) and co-precipitation of G (middle panel). The precipitated products were analyzed on SDS-PAGE under reducing conditions and then western blot analyzed with F (bottom panel) or G (top and middle panel) specific mouse mAbs. The western blot result of one of three independent experiments is shown in (B). The relative HeV-F binding ability of each HeV-G mutant is shown in comparison to that of WT HeV-G and normaliz.Alanine mutagenesis experiments support the model for HeV-G mediated HeV-F activation. However, it should be pointed out that our structure based observations were derivedusing a soluble form of the HeV-G glycoprotein and would not take into account potential long-range effects on the structure of G or its native tetrameric configuration because of the absence of the stalk, transmembrane domain and cytoplasmic tail. Finally, our studies document that targeted mutations, such as I588A, can be designed in the henipavirus attachment proteins, which appear to uncouple host cell/ephrin attachment and viral fusion initiation, providing important tools for additional functional and structural analysis of the precise molecular steps underlying the receptor triggering mechanism of henipavirus entry, and how such mutations in the G attachment glycoprotein accomplish this block. The structure-based models discussed above also shed light on the dynamic process of the virus-receptor attachment stage, as well as on the mechanism of the G glycoprotein initiated fusionpromotion step that facilitates F activation. The structures provide a clear basis to design further experiments for evaluation of models of henipavirus attachment and entry into host cells and to examine certain key steps in the paramyxovirus entry process in general. Further, the series of structure-based point mutations made in the HeV-G glycoprotein underscore the importance of the individual residues forming the ephrin binding interface, and indicate that both the crystallographically-observed hydrophobic and electrostatic interactions are crucial not only for cell attachment, but also for the subsequent membrane fusion and viral entry events. In addition, our structure-based mutagenesis experiments reveal three important and unique features of the HeV entry process: First, that single substitutions of interface resides affect more strongly the HeV-G binding and entry to ephrin-B3-expressing cells as compared to ephrin-B2-expressing cells, suggesting that the HeV-G/ephrin-B2 attachment is more robust and resistant to minor structural perturbations; Second, that that the stability of the HeV-G/ephrin association does not strongly correlate with the efficiency of viral entry, suggesting that, particularly for ephrin-B2expressing cells, viral attachment is not the rate limiting step in the viral entry process; And third, that it is possible to alter the HeVG/ephrin interface in a way that does not affect the overall stability and/or affinity of the association, but that does affect the efficiency of viral entry, in the context of henipavirus pseudovirions, supporting a model where subtle structural changes at the HeV-G/ephrin interface are relayed at a distance to trigger largeFigure 9. Interaction of HeV-G mutants with HeV-F. (A) HeV-G mutants were co expressed with S tagged HeV-F in HeLa-USU cells. Lysates were immunoprecipitated (IP) with rabbit polyclonal G-specific antiserum to evaluate total G expression (top panel) or S agarose to evaluate total F expression (bottom panel) and co-precipitation of G (middle panel). The precipitated products were analyzed on SDS-PAGE under reducing conditions and then western blot analyzed with F (bottom panel) or G (top and middle panel) specific mouse mAbs. The western blot result of one of three independent experiments is shown in (B). The relative HeV-F binding ability of each HeV-G mutant is shown in comparison to that of WT HeV-G and normaliz.
