Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin - PubMed (original) (raw)
Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin
B Schroth-Diez et al. J Virol. 1998 Jan.
Abstract
The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.
Figures
FIG. 1
Expression of HA and HA-derived chimeras in CV-1 cells. CV-1 transfected with wt HA (H/H/H), H/H/F, H/F/H, H/F/F, and H/HF/F (left gel) or with vaccinia virus (VAC), H/H/H, H/H/N, and H/H/R (right gel) were labeled with [35S]methionine/cysteine. Cell extracts were immunoprecipitated with antibody against influenza virus and subjected to SDS-PAGE (12% polyacrylamide gel) followed by fluorography (1 day). The positions of uncleaved HA0 and its two subunits, HA1 and HA2, in the gel are marked on the left.
FIG. 2
Histograms of the FACS analysis of CV-1 cells. The cells cultured in 35-mm dishes were infected with vTF7-3 vaccinia virus and transfected with plasmids encoding the respective protein as designated. After being labeled with fluorescent antibodies, the cells were treated as described in Materials and Methods and analyzed by FACS. VAC represents CV-1 cells infected with wt vaccinia virus (control); H/H/H, H/H/F, H/HF/F, H/F/F, H/F/H, H/H/N, and H/H/R represent for cells transfected with the respective constructs. Cell numbers (Counts) are plotted against fluorescence intensity (FL1-Height). About 50,000 were analyzed in each experiment, except for the H/F/F and H/F/H experiments in which about 20,000 cells were measured. The calculated transfection efficiency and mean fluorescence index are shown in Table 2.
FIG. 3
Fluorographs of doubly labeled RBCs bound to CV-1 cells expressing chimeric or wt HA proteins. Chimeric proteins mediate membrane mixing as well as cytoplasmic content mixing, as demonstrated by fluorescence microscopy. Doubly labeled (R18 and calcein) human RBCs were bound to monkey kidney cells expressing the respective protein as described in Materials and Methods. At 4 min after the pH was reduced to 5.0, R18 label was present in the membranes of the CV-1 cells, indicating the mixing of membrane lipids. The flow of calcein from the lumen of the bound RBCs into the cytoplasm of the CV-1 cells was observed as well. This reveals that pores which permit the transfer of aqueous compounds with a low molecular weight have been formed. All observations were taken at room temperature. (Top row) R18 fluorescence at pH 7.4; (second row) calcein fluorescence at pH 7.4; (third row) R18 fluorescence 4 min after the pH was reduced to 5.0; (bottom row) calcein fluorescence 4 min after the pH was reduced to 5.0; (left column) H/H/H; (second column) H/H/F; (third column) H/F/H; (right column) H/F/F.
FIG. 4
Fluorographs of doubly labeled RBCs bound to CV-1 cells expressing H/H/N or H/H/R proteins. Doubly labeled (R18 and calcein) human RBCs were bound to monkey kidney cells expressing H/H/N or H/H/R constructs at pH 7.4 (not shown). At 6 min after the pH was reduced to 5.0, R18 label was present in the membranes of the CV-1 cells, indicating the mixing of the membrane lipids. The flow of calcein from the lumen of the bound RBC into the cytoplasm of the CV-1 cell was observed as well. This reveals that pores which permit the transfer of aqueous compounds with low molecular weights have been formed. The experiments were performed at room temperature. (Top row) R18 fluorescence 6 min after the pH was reduced to 5.0; (bottom row) calcein fluorescence 6 min after the pH was reduced to 5.0; (left column) H/H/N; (right column) H/H/R.
FIG. 5
Kinetics of FDQ of R18-labeled RBCs bound to transfected CV-1 cells at different pHs. Typical time courses of the fusion of CV-1 cells expressing H/H/H or the H/F chimeric constructs with R18-labeled human RBCs at 37°C are shown. The cell-RBC suspension was added to the buffer in the cuvette at the respective pHs as indicated, and fusion monitoring started immediately (t = 0). At t = 590 s, Triton X-100 was added for infinite dilution of the fluorophore. The kinetics were normalized as described by Morris et al. (20). The fluorescence intensity at pH 7.4 was set to 0%, and fluorescence intensity after the addition of Triton X-100 was set to 100% FDQ (for details, see Materials and Methods). The results of two independent experiments with wt HA (H/H/H) are presented to show the experimental variability.
FIG. 6
Kinetics of the FDQ of R18-labeled RBCs bound to H/H/N- and H/H/R-transfected CV-1 cells at different pHs. Typical time courses of fusion of CV-1-cells expressing H/H/N and H/H/R chimeric constructs with R18-labeled human RBCs at 37°C are shown. The experimental procedures were the same as those described for Fig. 5 (note the different scaling). Curve A, H/H/N at pH 5.0; curve B, H/H/R at pH 5.0; curve C, H/H/N and H/H/R at pH 6.7.
FIG. 7
pH dependence of the maximal FDQ of R18-labeled erythrocytes bound to CV-1 cells expressing foreign protein. The kinetics of the FDQ of R18 at different pH values were recorded and normalized as described for Fig. 5. For each studied protein, the maximal FDQ values before the addition of Triton X-100 are plotted against the respective pH values. The inserted graph vac-control in panel B represents the control with CV-1 cells infected by vaccinia virus without any foreign DNA. For this measurement, labeled RBCs were bound to CV-1 cells via WGA. For all probes except the vac-control, the FDQ values were normalized to the respective FDQ maximum of the pH dependence. The maximal FDQ ranged between 30 and 60%.
References
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