Usion promotion activity of HeV-G, we next examined the impact of

Usion promotion activity of HeV-G, we next examined the impact of the HeV-G mutations on the efficiency of virus entry. Wild-type or the mutant HeV-G constructs were expressed together with HeV-F to generate a series of lentivirus-based, reporter gene encoding pseudovirions as described in the Materials and Anlotinib custom synthesis Methods [51]. These HeV glycoprotein-containing pseudovirions were then used to infect either ephrin-B2 or ephrin-B3 expressing target cells. As the structural data suggested, mutations in the interface residues had, in general, a significant effect on virus entry (Figure 8). Six of the 11 mutations (N402A, E501A, E505A, G506A, E533A and I588A) completely abrogated virus entry on ephrin-B2 expressing cells (Figure 8A), while one mutant (Y581A) a minor inhibitory effect. Interestingly, three of these six substitutions affected electrostatic, rather than hydrophobic HeV-G-ephrin interactions: the K103E501, and K113-E533 salt bridges, discussed above, and theHendra Virus Entry Mechanism Implied by StructureFigure 6. The ephrin-B2 W122 “latch”. Two rotameric W122 (ephrin-B2) conformations in the HeV-G/ephrin-B2 complex, both of which are distinct from the W122 conformation in unbound ephrin-B2. Ephrin-B2 is shown in silver in the unbound state, and in yellow or cyan in the two complexes with HeV-G (A). W122 transforms from the initial unbound conformation to an inter115103-85-0 biological activity mediate conformation upon binding to HeV-G due to steric and electrostatic constrains, then adopts its final conformation via stabilizing van der Waals interactions with HeV-G. W122 is shown as yellow sticks. G is shown as a surface colored according to its electrostatic potential (B). “Latch up” and “latch down” conformations of ephrin-B2-W122 mediate the association and dissociation of the HeV-G/ephrin-B2 complex. Ephrin-B2-W122 is shown in yellow, HeV-G – in grey (C). doi:10.1371/journal.pone.0048742.gW122-E505 “latch” interaction which positions the W122 side chain (Figure 6). Two mutations (V401A and Q490A) exhibited a notable enhancing effect, which is possibly due, in the case of Q490A, to increase in the hydrophobicity of the binding pockets for the ephrin G-H loop, and, in case of V401A, to facilitate of the transition to the more stable “latch down” conformation of the W122 side chain (Figure 6). The W504A and Q559A mutations had a modest yet reproducible enhancing effect on pseudotyped virus entry in comparison to wild-type G (Figure 8A). To further validate these observations, the same series of HeV pseudovirions where used to infect ephrin-B3 expressing cells, and here the effects on virus entry were more profound. Furthermore, in addition to the same G residues showing inhibition upon mutation to alanine, three other amino acid substitutions (Q490A, W504A and Y581A) also completely or 1527786 significantly abrogated virus entry in ephrin-B3 expressing cells (Figure 8B). In addition,2 of 3 changes (V401A and Q559A) that enhanced the virus entry on ephrin-B2 expressing cells also enhanced virus entry on ephrinB3 expressing cells. Together, these data reveal an overall similar functional importance of the interface residues within HeV-G for both ephrin-B2 and -B3 receptor usage and also support our W122 latch model. To confirm that impaired function of the HeVG mutants was not due to a lack of glycoprotein incorporation into the viral pseudotypes, equivalent amounts of each virus pseudotype preparation used in the experiment shown in Figure 8A and 8B were lysed a.Usion promotion activity of HeV-G, we next examined the impact of the HeV-G mutations on the efficiency of virus entry. Wild-type or the mutant HeV-G constructs were expressed together with HeV-F to generate a series of lentivirus-based, reporter gene encoding pseudovirions as described in the Materials and Methods [51]. These HeV glycoprotein-containing pseudovirions were then used to infect either ephrin-B2 or ephrin-B3 expressing target cells. As the structural data suggested, mutations in the interface residues had, in general, a significant effect on virus entry (Figure 8). Six of the 11 mutations (N402A, E501A, E505A, G506A, E533A and I588A) completely abrogated virus entry on ephrin-B2 expressing cells (Figure 8A), while one mutant (Y581A) a minor inhibitory effect. Interestingly, three of these six substitutions affected electrostatic, rather than hydrophobic HeV-G-ephrin interactions: the K103E501, and K113-E533 salt bridges, discussed above, and theHendra Virus Entry Mechanism Implied by StructureFigure 6. The ephrin-B2 W122 “latch”. Two rotameric W122 (ephrin-B2) conformations in the HeV-G/ephrin-B2 complex, both of which are distinct from the W122 conformation in unbound ephrin-B2. Ephrin-B2 is shown in silver in the unbound state, and in yellow or cyan in the two complexes with HeV-G (A). W122 transforms from the initial unbound conformation to an intermediate conformation upon binding to HeV-G due to steric and electrostatic constrains, then adopts its final conformation via stabilizing van der Waals interactions with HeV-G. W122 is shown as yellow sticks. G is shown as a surface colored according to its electrostatic potential (B). “Latch up” and “latch down” conformations of ephrin-B2-W122 mediate the association and dissociation of the HeV-G/ephrin-B2 complex. Ephrin-B2-W122 is shown in yellow, HeV-G – in grey (C). doi:10.1371/journal.pone.0048742.gW122-E505 “latch” interaction which positions the W122 side chain (Figure 6). Two mutations (V401A and Q490A) exhibited a notable enhancing effect, which is possibly due, in the case of Q490A, to increase in the hydrophobicity of the binding pockets for the ephrin G-H loop, and, in case of V401A, to facilitate of the transition to the more stable “latch down” conformation of the W122 side chain (Figure 6). The W504A and Q559A mutations had a modest yet reproducible enhancing effect on pseudotyped virus entry in comparison to wild-type G (Figure 8A). To further validate these observations, the same series of HeV pseudovirions where used to infect ephrin-B3 expressing cells, and here the effects on virus entry were more profound. Furthermore, in addition to the same G residues showing inhibition upon mutation to alanine, three other amino acid substitutions (Q490A, W504A and Y581A) also completely or 1527786 significantly abrogated virus entry in ephrin-B3 expressing cells (Figure 8B). In addition,2 of 3 changes (V401A and Q559A) that enhanced the virus entry on ephrin-B2 expressing cells also enhanced virus entry on ephrinB3 expressing cells. Together, these data reveal an overall similar functional importance of the interface residues within HeV-G for both ephrin-B2 and -B3 receptor usage and also support our W122 latch model. To confirm that impaired function of the HeVG mutants was not due to a lack of glycoprotein incorporation into the viral pseudotypes, equivalent amounts of each virus pseudotype preparation used in the experiment shown in Figure 8A and 8B were lysed a.