Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies - PubMed (original) (raw)
Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies
Karin Stiasny et al. J Virol. 2007 Oct.
Abstract
In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.
Figures
FIG. 1.
Schematic model of the flavivirus fusion process (A to E) and ribbon diagrams of the sE prefusion dimer (F and G) and postfusion trimer (H) of TBEV. E protein DI, red; E DII, yellow; E DIII, blue; FP, orange; stem, purple; transmembrane anchor, green; viral membrane, blue; target membrane, gray. (A) Metastable E dimer on the surface of native virions. (B) Low pH-induced dissociation of the dimer and insertion of the FP into the target membrane. (C) Relocation of DIII leading to hairpin formation, trimerization, and “zippering” of the stem along the body of the trimer. (D) Hemifusion intermediate. Only the contacting membrane leaflets are fused. (E) Fusion pore formation. In the final postfusion conformation, the FPs and the membrane anchors are juxtaposed in the fused membrane. (F) Side view and (G) top view of the sE dimer. (H) Side view of the sE trimer. The gray balls show the positions of mutations that affected binding of MAbs. The position of the carboxy terminus of sE is indicated by a purple arrow and labeled COO−.
FIG. 2.
Low pH-induced fusion of pyrene-labeled TBEV in the absence (No mab) or presence of MAbs in a pyrene excimer fusion assay. Pyrene-labeled TBEV virions were preincubated with each of the MAbs overnight at 4°C, liposomes were added, the mixture was acidified, and the change in pyrene excimer fluorescence was monitored continuously for 60 s. The fusion curves were averaged, and the data shown are from at least three independent experiments. (A) Abolition of fusion (MAbs A3, A4, IC3). (B) No significant effect on fusion (MAbs B2, B3). (C) Reduction of the extent of fusion (MAbs A1, A2, A5). (D) Reduction of both rate and extent of fusion (MAbs IE3, i2, B1, B4).
FIG. 3.
Low pH-induced coflotation of TBEV with liposomes in the absence or presence of MAbs. TBEV virions were preincubated with each of the MAbs overnight at 4°C, liposomes were added, and the mixture was acidified, back-neutralized, and then subjected to centrifugation in sucrose step gradients. (A) Representative examples of coflotation experiments. From left, panel 1, coflotation without MAb (No mab) at pH 5.4 (solid line) and pH 7.4 (dotted line). Panels 2 to 4, coflotation in the presence of MAbs at pH 5.4. B2 (panel 2; no effect), A3 (panel 3; strong effect), and A2 (panel 4; intermediate effect). (B) Results of coflotation experiments with each of the MAbs, expressed as percentages of E protein bound to liposomes at pH 5.4 in comparison to the control without a MAb. The data are the averages from at least two independent experiments; the error bars represent the standard errors of the means.
FIG. 4.
(A) Blocking ELISA in the absence of detergent with native virions and MAbs A1, A2, A4, B2, and B4 as described previously (27). A predetermined fixed dilution of the respective MAb was incubated with decreasing concentrations of native TBEV. The mixture was then added to microtiter plates that had been coated with purified virus at a concentration of 0.5 μg/ml, a procedure that leads to the exposure of the FP loop, allowing its reaction with FP-specific MAbs (27). Antibody that was not blocked by the antigen in solution bound to the solid-phase antigen and was detected using a peroxidase-labeled rabbit anti-mouse immunoglobulin G (27). Results are expressed as percentages of the absorbance value obtained with each MAb in the absence of a blocking antigen. The data are representative of results of at least two independent experiments. (B) Four-layer ELISA with TBEV and MAb A1 to analyze the transient exposure of the A1 binding site upon acidification. Native TBEV in phosphate-buffered saline (pH 7.4; protein concentration, 0.5 μg/ml) was captured by polyclonal anti-TBEV immunoglobulin G for 1 h at 37°C as described previously (27). A1 epitope exposure was tested with the following combinations of pH and biotin-labeled MAb: column A, A1 was added in phosphate-buffered saline (pH 7.4); column B, A1 was added in MES buffer (pH 5.5; 50 mM MES, 100 mM NaCl); column C, the captured virus was exposed to MES buffer (pH 5.5) for 10 min followed by MAb A1 in the same buffer. After incubation for 1 h at 37°C, bound antibodies were detected by using streptavidin-peroxidase (Sigma-Aldrich). The data are the averages from five independent experiments performed in duplicate, and the error bars represent the standard errors of the means.
References
- Burke, D. S., and T. P. Monath. 2001. Flaviviruses, p. 1043-1126. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
- Corver, J., A. Ortiz, S. L. Allison, J. Schalich, F. X. Heinz, and J. Wilschut. 2000. Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system. Virology 269:37-46. - PubMed
- Crowe, J. E., Jr., R. O. Suara, S. Brock, N. Kallewaard, F. House, and J. H. Weitkamp. 2001. Genetic and structural determinants of virus neutralizing antibodies. Immunol. Res. 23:135-145. - PubMed
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