Identification of specific histidines as pH sensors in flavivirus membrane fusion

Abstract

The flavivirus membrane fusion machinery, like that of many other enveloped viruses, is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. It has been hypothesized that conserved histidines in the class II fusion protein E of these viruses function as molecular switches and, by their protonation, control the fusion process. Using the mutational analysis of recombinant subviral particles of tick-borne encephalitis virus, we provide direct experimental evidence that the initiation of fusion is crucially dependent on the protonation of one of the conserved histidines (His323) at the interface between domains I and III of E, leading to the dissolution of domain interactions and to the exposure of the fusion peptide. Conserved histidines located outside this critical interface were found to be completely dispensable for triggering fusion.

Figure 1.

Summary of the organization of flavivirus particles, the three-dimensional structures of the flavivirus envelope protein E, and a model of flavivirus membrane fusion. (A) Schematic diagram of a flavivirus particle in its immature (prM-containing) and mature form after proteolytic cleavage of prM. (B) Schematic of the prefusion E dimer including ribbon diagrams of the TBEV sE ectodomain (top and side view) and those parts for which the atomic structure is not known (stem and anchor). (C) Ribbon diagram of the postfusion TBEV sE trimer (side view). The positions of the histidines conserved in all flavivirus E proteins are indicated by gray balls. (D) Schematic of the proposed flavivirus fusion mechanism showing different steps of the fusion process. (step 1) Metastable E dimer in mature virions. (step 2) Dissociation of the E dimers at acidic pH, outward projection of E monomers, and interaction of the FP with the target membrane. (step 3) Trimerization, DIII relocation, and “zipping up” of the stem. (step 4) Formation of the postfusion trimer and opening of the fusion pore. Red, DI; yellow, DII; blue, DIII; orange, FP; purple, stem (linker between DIII and the transmembrane anchors); gray, transmembrane anchors.

Figure 2.

Fusion activity of pyrene-labeled WT and mutant RSPs with liposomes at acidic pH. (A) Kinetic fusion curves of RSP WT (red) and the mutant RSPs H323A (orange) and H248N-H287A (blue) at pH 5.4. (B) Extent of fusion after 60 s of mutant RSPs relative to that of the WT (set at 100%). The experiments with those mutants that were significantly different from the WT were performed at least twice and the error bars represent the standard errors of the means.

Figure 3.

Acidic pH–induced FP exposure as measured by the binding of the FP-specific mAb A1. Results are expressed as a percentage of the maximal reactivity of A1 obtained with the WT at pH 5.4. The data are the means of three independent experiments performed in duplicate; the error bars represent the standard errors of the means.

Figure 4.

Acidic pH–induced coflotation of WT and mutant RSPs with liposomes. RSPs were incubated with liposomes at pH 5.4 (solid lines) and 8.0 (dotted lines), back-neutralized, and then subjected to centrifugation in sucrose step gradients. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA. The top fractions containing RSPs coflotated with the liposomes are indicated by a bracket. (A–C) Representative examples of the analysis of the step gradients obtained with WT (A) and the fusion-negative mutants H323A (B) and H248N-H287A (C). The inset in C shows the fluorescence spectrum of the coflotated fractions of the mutant (gray) relative to those of the WT (black) from experiments using pyrene-labeled RSPs. AU, arbitrary units. (D) Extent of acidic pH–induced coflotation of E with liposomes obtained with mutant RSPs relative to the WT (set at 100%). The data are the means from at least two independent experiments; the error bars represent the standard errors of the means.

Figure 5.

Analysis of acidic pH–induced trimer formation with WT and mutant RSPs by rate zonal gradient centrifugation. (A–D) RSPs were incubated for 10 min at pH 5.4 (solid line) or 8.0 (dotted line), back-neutralized, solubilized with 1% Triton X-100, and analyzed by sedimentation in 7–20% sucrose gradients containing 0.1% Triton X-100. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA. The sedimentation direction is from left to right. Peak of the E dimer, fraction 8; peak of the E trimer, fraction 10.

Figure 6.

pH threshold of E trimer formation of RSP WT and mutants. Extent and pH dependence of acidic pH–induced E trimer formation with WT and mutant RSPs as determined by rate zonal sucrose density centrifugation. Results are expressed as a percentage of E found in the trimer peak fractions relative to the total amount of E in the gradient.

Figure 7.

Analysis of the stability of acidic pH–induced E trimers of WT and selected mutant RSPs. Acidic pH–induced trimers of WT (red) and mutant RSPs (green, H248A; orange, H323A; blue, H248A-H287N) were exposed to 70°C and subjected to rate zonal sucrose density gradient centrifugation. The sedimentation direction is from left to right. The pellet (P) was resuspended in 0.6 ml corresponding to the volume of a single fraction.

Figure 8.

Intramolecular interactions of DIII in the pre- and postfusion structures of sE. (A) Ribbon diagrams of the TBEV sE dimer and the details of the DI–DIII interface in the prefusion conformation with the central salt bridge between Arg9 and Glu373. (B) Ribbon diagrams of the TBEV sE trimer and the details of DIII in its postfusion conformation revealing the possible salt bridge between His323 and Glu373.