Nd the relative levels of the G and F glycoproteins were measured by SDS-PAGE and Western blot as previously reported [51] (Figure 8C). This analysis revealed that most of the HeV-G mutants were incorporated into their respective pseudotyped virus preparations at levels equivalent to or greater than wild-type HeV-G, with exception of the N402A and E533A mutants. The HeV-F glycoprotein was incorporated at levelsHendra Virus Entry Mechanism Implied by StructureFigure 8. Effect of structure-based HeV-G mutations on viral entry. Luciferase-encoding HIV-1 based pseudovirus stocks were prepared in 293T cells using wild-type (WT) or alanine substitution mutants of HeV-G with the HeV-F by expression plasmid transfection together with pNL4-3-Luc-E-R+ as described in Methods. Each pseudovirus stock preparation was analyzed by p24 quantification and equal amounts of virus particles were used to infect target cells, either HelaUSU cells expressing BIBS39 ephrin-B2 (A) or ephrin-B3 (B), and performed in KS 176 custom synthesis triplicate. Cells were incubated for 48 hr following infection and processed for luciferase activity quantification using a Centro LB 960 Microplate Luminometer (Berthold Technologies). This experiment was repeated six times and a representative experiment is shown. (C) Incorporation of the HeV F and wild-type and mutant G glycoproteins into pseudovirus particles was assessed by Western blot of lysates prepared from p24 normalized amounts of the same purified virus particles used in Panels A and B. HeV G was detected with a crossreactive polyclonal mouse antiserum to HeV G and HeV F was detected with a rabbit polyclonal F1 specific antiserum as described in the Methods. doi:10.1371/journal.pone.0048742.gFigure 7. Expression and receptor binding activity of structurebased HeV-G mutations. The various alanine substitution mutants or wild-type (WT) HeV-G were transiently expressed in the absence (A) or presence (B) of HeV-F in HeLa-USU cells. Cell lysates were prepared and equal amounts were co-precipitated with recombinant ephrin-B2/Fc or ephrin-B3/Fc, or directly immunoprecipitated with polyclonal G-specific antibodies (control), followed by protein G Sepharose beads. The precipitated samples were processed and analyzed by 4 to 20 gradient SDS-PAGE and Western blotting with HeV- G-specific antiserum. This experiment was repeated three times and one representative experiment is shown. doi:10.1371/journal.pone.0048742.gequivalent to or greater than wild-type HeV F in all pseudotyped particle preparations (Figure 8C). Importantly, the entry inhibitory effects of the majority of the HeV-G mutations that either completely abrogated or inhibited virus entry in ephrin-B2 or ephrin-B3 expressing cells (E501A, E505A, G506A, I588A and Y581A), as well as the HeV-G mutants Q490A, W504A whichblocked entry on ephrin-B3 expressing cells, were not a result of poor incorporation of the glycoproteins into the pseudovirions. Thus, most of the mutations, which disrupted HeV entry in ephrin-B2 expressing cells in the context of a pseudotyped virus particle (E501A, E505A, G506A, Y581A, I588A), were not doing so because the mutant G glycoprotein was poorly incorporated into the particles, nor did they have a defect in their ability to bind the ephrin-B2 receptor. The minor difference in the behavior of the W504A substitution in HeV-G, which destabilizes the HeVG/ephrin-B2 complex, from that of the equivalent mutation in NiV-G, which does not seem to affect the NiV-G/ephrin-B2 bin.Nd the relative levels of the G and F glycoproteins were measured by SDS-PAGE and Western blot as previously reported [51] (Figure 8C). This analysis revealed that most of the HeV-G mutants were incorporated into their respective pseudotyped virus preparations at levels equivalent to or greater than wild-type HeV-G, with exception of the N402A and E533A mutants. The HeV-F glycoprotein was incorporated at levelsHendra Virus Entry Mechanism Implied by StructureFigure 8. Effect of structure-based HeV-G mutations on viral entry. Luciferase-encoding HIV-1 based pseudovirus stocks were prepared in 293T cells using wild-type (WT) or alanine substitution mutants of HeV-G with the HeV-F by expression plasmid transfection together with pNL4-3-Luc-E-R+ as described in Methods. Each pseudovirus stock preparation was analyzed by p24 quantification and equal amounts of virus particles were used to infect target cells, either HelaUSU cells expressing ephrin-B2 (A) or ephrin-B3 (B), and performed in triplicate. Cells were incubated for 48 hr following infection and processed for luciferase activity quantification using a Centro LB 960 Microplate Luminometer (Berthold Technologies). This experiment was repeated six times and a representative experiment is shown. (C) Incorporation of the HeV F and wild-type and mutant G glycoproteins into pseudovirus particles was assessed by Western blot of lysates prepared from p24 normalized amounts of the same purified virus particles used in Panels A and B. HeV G was detected with a crossreactive polyclonal mouse antiserum to HeV G and HeV F was detected with a rabbit polyclonal F1 specific antiserum as described in the Methods. doi:10.1371/journal.pone.0048742.gFigure 7. Expression and receptor binding activity of structurebased HeV-G mutations. The various alanine substitution mutants or wild-type (WT) HeV-G were transiently expressed in the absence (A) or presence (B) of HeV-F in HeLa-USU cells. Cell lysates were prepared and equal amounts were co-precipitated with recombinant ephrin-B2/Fc or ephrin-B3/Fc, or directly immunoprecipitated with polyclonal G-specific antibodies (control), followed by protein G Sepharose beads. The precipitated samples were processed and analyzed by 4 to 20 gradient SDS-PAGE and Western blotting with HeV- G-specific antiserum. This experiment was repeated three times and one representative experiment is shown. doi:10.1371/journal.pone.0048742.gequivalent to or greater than wild-type HeV F in all pseudotyped particle preparations (Figure 8C). Importantly, the entry inhibitory effects of the majority of the HeV-G mutations that either completely abrogated or inhibited virus entry in ephrin-B2 or ephrin-B3 expressing cells (E501A, E505A, G506A, I588A and Y581A), as well as the HeV-G mutants Q490A, W504A whichblocked entry on ephrin-B3 expressing cells, were not a result of poor incorporation of the glycoproteins into the pseudovirions. Thus, most of the mutations, which disrupted HeV entry in ephrin-B2 expressing cells in the context of a pseudotyped virus particle (E501A, E505A, G506A, Y581A, I588A), were not doing so because the mutant G glycoprotein was poorly incorporated into the particles, nor did they have a defect in their ability to bind the ephrin-B2 receptor. The minor difference in the behavior of the W504A substitution in HeV-G, which destabilizes the HeVG/ephrin-B2 complex, from that of the equivalent mutation in NiV-G, which does not seem to affect the NiV-G/ephrin-B2 bin.
