Figure 1.
Structures of the conformations assumed by HA during the virus life cycle.
Trimeric HA molecule is shown as a ribbon diagram, with the HA1 subunit colored in blue, the HA2 subunit colored in red, and residues corresponding to the fusion peptide colored in yellow. (A) The site within the trimeric HA molecule that acts as a substrate for activating protease (i.e. the cleavage loop) is denoted with an arrow. (B) Upon proteolytic activation within the cleavage loop, the N-terminus of the newly generated HA2 subunit is relocated to the trimer interior where it forms extensive contacts with a network of residues within the fusion peptide pocket. This forms the metastable conformation that is primed for fusion. (C) Upon exposure to low pH, the HA undergoes a series of conformational changes that result in the extrusion of the fusion peptide for mediating membrane fusion between the virion and endosomal membranes.
Table 1.
Origin of HA and NA proteins examined and HA cleavage site sequence.
Figure 2.
Analysis of trypsin-mediated cleavage-activation of HA in the absence and presence of NA.
(A) BHK cells were transfected with 1.0 µg HA plasmid or co-transfected with 1.0 µg HA plasmid and 0.1 µg NA plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Where indicated, radiolabeled, HA-expressing cell monolayers were treated with 5 µg/ml TPCK-Trypsin for 15 min. Total cellular HA protein was immunoprecipitated with a HA-specific antibody and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. Representative images of gels are shown with the HA subtype denoted at the top of each gel and the identity of each band is denoted on the left. (B) Quantitation of trypsin-mediated cleavage of HA in the absence of NA. Quantitative analysis was performed on three independent experiments. For each HA, the intensity of the bands corresponding to HA0 and HA2 were normalized to their respective methionine content and the percent cleavage (mean ± standard deviation) is expressed as a percentage of the total cellular HA, by the equation (HA2/(HA2+HA0))×100%. * denotes no detectable cleavage. (C) Quantitation of surface-expressed HA. BHK cells were transfected with 1.0 µg HA plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Cell surface proteins were then biotinylated and total HA protein was immunoprecipitated with a HA-specific antibody, followed by immunoprecipitation with streptavidin, and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. Quantitative analysis of three independent experiments is shown. The percentage of surface-expressed HA was determined by dividing the amount of surface-expressed HA by the total HA. (D) Quantitation of trypsin-mediated cleavage of HA in the presence of NA. Quantitative analysis was performed as described in (B). * denotes no detectable cleavage.
Figure 3.
Analysis of proteolytic cleavage of H11 HA and surface expression of H13 and H16 HAs.
(A) BHK cells were transfected with 1.0 µg H11 HA plasmid or co-transfected with 1.0 µg H11 HA plasmid and 0.1 µg N6 NA plasmid. Experimental details are identical to those stated in Figure 2A, except that during the chase period media either lacking serum or containing 10% fetal bovine serum was used. The conditions assayed for the samples in each lane are denoted at the top of the gel and the identity of each band is denoted on the left. (B) Quantitation of H11 HA cleavage in the presence and absence of NA, the presence and absence of 10% fetal bovine serum, and in the presence and absence of trypsin. Quantitative analysis was performed on three independent experiments. For each HA, the intensity of the bands corresponding to HA0 and HA2 were normalized to their respective methionine content and the percent cleavage (mean ± standard deviation) is expressed as a percentage of the total cellular HA, by the equation (HA2/(HA2+HA0))×100%. (C) BHK cells were transfected with 1.0 µg H13 or H16 HA plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Cell surface proteins were then biotinylated and total HA protein was immunoprecipitated with a HA-specific antibody, followed by immunoprecipitation with streptavidin, and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. The HA subtype is denoted at the top of each gel and the identity of each band is denoted on the left. T = Total HA, S = Surface expressed HA. (D) BHK, Vero, or DF-1 cells were transfected with 1.0 µg of H13 HA plasmid. Experimental details are the same as described in 2A.
Figure 4.
Effect of co-expressing NA on cell surface expression of HA.
(A) BHK cells were transfected with 1.0 µg H13 plasmid only, or co-transfected with 0.1 µg, 0.5 µg, or 1.0 µg N6 plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Cell surface proteins were then biotinylated and total HA protein was immunoprecipitated with a HA-specific antibody, followed by immunoprecipitation with streptavidin, and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. The amount of NA plasmid is denoted at the top of each gel and the migration of HA0 is denoted to the left of the gel. T = Total HA, S = Surface expressed HA. The graph to the right of the gel shows the quantitative analysis of three independent experiments. The percentage of surface-expressed HA was determined by dividing the amount of surface-expressed HA by the total HA. (B) Experimental details are the same as in A, except that BHK or Vero cells were transfected with 1.0 µg H3, H5, H7, Japan, or Aichi HA plasmid only or in combination with 0.1 µg of cognate NA plasmid. The data shown represents the quantitative analysis of three independent experiments. Quantitation was performed exactly as stated in (A).
Figure 5.
Analysis of HA cleavage by human serine proteases, HAT and TMPRSS2.
(A) BHK cells were transfected with 1.0 µg HA plasmid and 0.25 µg of either HAT or TMPRSS2 expression plasmid. At 16 hours post transfection, cells were starved for methionine and cysteine, and were pulse labeled with 25 µCi [35S]-methionine for 15 min, followed by a 3 hr chase period. Total HA protein was immunoprecipitated with a HA-specific antibody and resolved on 12% SDS-PAGs. The gels were fixed and dried, and radiolabeled proteins were visualized by phosphorimaging. The identity of each HA is denoted above the gel and the migration of HA0, HA1, and HA2 is denoted to the left of the gel. (B) Quantitation of HAT-mediated cleavage of HA. Quantitative analysis was performed on three independent experiments. For each HA, the intensity of the bands corresponding to HA0 and HA2 were normalized to their methionine content and the percent cleavage (mean ± standard deviation) is expressed as a percentage of the total cellular HA, by the equation (HA2/(HA2+HA0))×100%. * denotes no detectable cleavage. (C) Quantitation of TMPRSS2-mediated cleavage of HA. Quantitative analysis was performed on three independent experiments, as described in (B). The percent cleavage shown for the H5 and H5VN HAs was determined by subtracting the percent cleavage observed in the absence of trypsin from the percent cleavage observed in the presence of TMPRSS2.
Figure 6.
The pH of fusion for a select representative group of HA proteins.
(A) Photomicrographs of syncytia formation assay. BHK cells were transfected with 1.0 µg HA plasmid. At 16 hrs post transfection, HA-expressing BHK cells were treated with TPCK-trypsin (5 µg/ml), followed by treatment with pH-adjusted PBS in 0.1 pH unit increments, neutralized, and incubated at 37°C for 2 hr. Cells were then washed with PBS, fixed, stained, and imaged. Photomicrographs corresponding to the last pH at which syncytia were observed and 0.1-pH unit higher are shown. (B) Luciferase reporter gene assay for fusion. Vero cells were co-transfected with 1.0 µg HA plasmid and 1.0 µg T7-Luciferase plasmid. At 16 hrs post transfection, HA-expressing Vero cells were treated with TPCK-trypsin (5 µg/ml), followed by treatment with C. soybean trypsin inhibitor (20 µg/ml). HA-expressing Vero cells were overlaid with BSR-T7/5 target cells that constitutively express T7 RNA polymerase. The two cell populations were incubated for 1 hr, at which time cell monolayers were pulsed with pH-adjusted PBS in 0.2 pH unit increments at the indicated pH for 5 min to trigger fusion, and then were neutralized. Cells were incubated for 6 hrs at 37°C to allow for cell-to-cell fusion to occur, which would mediate transfer of the T7-luciferase plasmid and expression of firefly luciferase. Luminescence was measured as an indicator of membrane fusion in cell lysates. The graphs show the mean background-adjusted relative luminescence (± standard deviation) as a function of pH obtained from three independent experiments.
Figure 7.
Summary of the pH of fusion obtained from the syncytium and luciferase assays for all subtypes examined.
(*) indicates no fusion was detected.
Table 2.
Summary of proteolytic cleavage and fusion data for all HAs examined.
Figure 8.
HA-mediated fusion in the absence of trypsin, the presence of trypsin, or the presence of TMPRSS2, as measured using the luciferase reporter gene fusion assay.
Vero cells were transfected with 1.0 µg HA plasmid, 1.0 µg T7 luciferase plasmid, and, where indicated, 0.25 µg TMPRSS2 plasmid. At 16 hours post transfection, HA-expressing Vero cells were either left untreated or treated with TPCK-trypsin (5 µg/ml), and overlaid with BSR-T7/5 target cells. Cells were then treated with PBS or PBS that had been pH adjusted to 5.0 with citric acid, neutralized, and incubated at 37°C for 6 hr to allow for cell-to-cell fusion to occur. Cell populations were then harvested and the luciferase activity resulting from the fused cell populations was quantified and is expressed as the mean ± standard deviation of relative luminescence units (RLU).