A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype - PubMed (original) (raw)
A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype
H Qiao et al. Mol Biol Cell. 1999 Aug.
Free PMC article
Abstract
We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.
Figures
Figure 1
Cell surface expression and proteolytic processing of WT and mutant HAs. Stable NIH 3T3 cells expressing WT and mutant HAs were treated with NaButyrate to enhance HA expression. The cells were then labeled with the membrane impermeant reagent NHS-LC-biotin, treated with either trypsin (to cleave HA0) or chymotrypsin (HA0 control), and lysed. Equal amounts of protein from each cell lysate were precipitated with the Site A mAb, and the immune complexes were separated by reducing SDS-PAGE. The gel was transferred to nitrocellulose, probed with streptavidin-HRP, and developed by enhanced chemiluminescence.
Figure 2
Proteinase K sensitivity of WT and mutant HAs. Cells expressing WT and mutant HAs were metabolically labeled with 35[S]-TransLabel, treated with 8 μg/ml trypsin for 6 min at RT, and then incubated at the indicated pH for 15 min at 37°C, reneutralized, and lysed in an NP-40 cell lysis buffer. Cell lysates were then digested with 0.2 mg/ml proteinase K for 30 min at 37°C. The proteins were immunoprecipitated with the Site A mAb, resolved by SDS-PAGE, and subjected to phosphorimager analysis.
Figure 3
Fusion activity of WT and mutant HAs: lipid mixing. COS7 cells transfected with plasmids encoding WT and mutant HAs were treated with neuraminidase (0.2 mg/ml) and either 5 μg/ml trypsin or chymotrypsin for 6 min at RT. R18-labeled RBCs (0.05%) were bound to the cells for 25 min at RT. After unbound RBCs were removed, the cells were incubated in fusion buffer, pH 5.0, for 2 min at 37°C, reneutralized, and observed with a fluorescence microscope.
Figure 4
Fusion activity of WT and mutant HAs by the R18 fluorescence dequenching assay. COS7 cells were transfected with plasmids encoding WT or mutant HAs, treated with neuraminidase and trypsin (or chymotrypsin), and then incubated with R18-labeled RBCs. The RBC–cell complexes were removed from the dish and placed in a cuvette containing fusion buffer, pH 7.0, in a fluorimeter. After attaining a baseline, the pH of the solution was lowered to 5.0 by injecting a predetermined amount of 1N citric acid, and the fluorescence was recorded. After lysis of the cells, the percentage of fluorescence dequenching was calculated, and the data were plotted with respect to time.
Figure 5
Comparison of lipid and content mixing fusion activity of WT HA and the Ala and Ser mutants. COS7 cells were transfected with plasmids (pSM) encoding WT, Ala, and Ser mutant HAs, and treated with neuraminidase and trypsin (T) or chymotrypsin (C) as described in the legend to Figure 3. The cells were incubated with RBCs labeled with both R18 (red) and Calcein AM (green), incubated in pH 5.0 fusion buffer for 2 min at 37°C, reneutralized, and observed with a confocal microscope.
Figure 6
Fusion activity of overexpressed Ser HA and Val HA. COS7 cells were transfected with 1× WT HA, 5× Ser HA, or 5× Val HA cDNA using the Mirus Transit reagent and processed for fusion with RBCs labeled with both R18 and calcein.
Figure 7
Simultaneous electrical recording of fusion pores and video fluorescence measurements of R18 transfer for RBC fusion to WT HA-expressing COS7 cells. (A) The pH of the solution was lowered at 0 s. The opening of a fusion pore between a WT HA-expressing cell and an RBC was marked by a spike in _Y_DC (at ∼17 s) and a simultaneous increment in _Y_90. _Y_DC returned nearly to baseline after the spike, showing that both the COS7 and RBC membrane conductances remained small. The fusion pore conductance (_G_p, bottom trace) was calculated from the _Y_90 trace (see MATERIALS AND METHODS). With enlargement of the fusion pore, the voltage applied across the COS7 cell membrane increasingly penetrated into the RBC, and _Y_90 was observed to increase. The voltage fully penetrated into the RBC once the pore enlarged sufficiently, and hence the precise value of _G_p (Figure 7A) became less certain, calculated as a “noisy” _G_p, once the _Y_90 neared saturation. (B) The time-dependent fluorescence intensities shown in C were fit with two straight lines, a constant fluorescence followed by a linearly increasing fluorescence. Their intersection determined the lag time from acidification until the onset of R18 spread (here 25.8 s, shown by arrowhead in A and B). (C) To temporally correlate the onset of membrane dye redistribution with conductance increases, for each experiment an ROI was selected for a COS7 cell (white contours), and the average intensity of fluorescence of this region, adjacent to a bound RBC, was determined on a frame-by-frame basis. The fluorescence images shown were obtained by averaging eight sequential video frames. The four images represent (from left to right) fluorescence patterns before dye spread, soon after dye spread began, after complete redistribution of R18, and the bright-field image at the conclusion of the experiment. Times after reducing the pH are indicated on all images and graphs.
Figure 8
Electrical and video fluorescence microscopy recordings for Ser HA-expressing cells induced to hemifuse to RBCs. (A) An experiment in which electrical changes were not detected for more than 5 min. (B) The onset of R18 spread was observed at 42.9 s. (C) Fluorescence and bright-field images for the experiment shown electrically in A. For details of quantifying dye spread, see legend to Figure 7. (D) Electrical profile of mild leakage for Ser HA-expressing cells hemifusing to RBCs. After pH was lowered, _Y_DC increased and fluctuated and the baseline of _Y_90 became appreciably more noisy, but the average value of _Y_90 did not change. A change in _Y_DC without a concurrent change in _Y_90 is the hallmark of membrane leaks, rather than fusion pore formation. R18 spread was observed (vertical arrowhead at 47.5 s) without pore formation. With time, the DC conductance became large (horizontal arrow), exceeding 50 nS. This increase in _Y_DC resulted in an excessively noisy _Y_90 trace, eliminating the ability to detect whether fusion pores formed at later times. RBCs tended to swell (increased size as time progressed can be seen in fluorescent images) and could even lyse (loss of contrast in bright-field images). RBC swelling is characteristic of leaks. We did not observe any electrical activity or dye spread when either WT HA or Ser HA were not activated by cleavage with trypsin (our unpublished results).
Figure 9
Chlorpromazine (CPZ) induces transfer of aqueous dye (CF) from RBCs to hemifused Ser HA-expressing cells. For each condition the ratio of COS7 cells stained with CF to those labeled by R18 was determined. In control experiments, after fusion was triggered by incubation for 2 min in a pH 5.0 solution at 37°C, and then incubated in PBS (supplemented with 20 mM raffinose) for 5 min at RT, virtually all WT HA-expressing cells (WT, first bar) were stained with both an aqueous dye (CF) and a membrane dye (R18). In contrast, CF spread into Ser HA-expressing cells was rarely observed, despite efficient R18 redistribution (second bar). These cells were exposed for 1 min at RT to either 0.05 mM (third bar) or 0.4 mM (fourth bar) CPZ, and then the CPZ-containing solution was replaced by PBS with raffinose. Error bars show the SE for 4–16 independent experiments.
References
- Carrasco L, Otero MJ, Castrillo JL. Modification of membrane permeability by animal viruses. Pharmacol Ther. 1989;40:171–212. - PubMed
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