Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus - PubMed (original) (raw)
Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus
K Stiasny et al. J Virol. 2001 Aug.
Abstract
The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.
Figures
FIG. 1
Thermal stability of E rosettes containing native E dimers (open symbols) or E trimers obtained by low-pH-treatment (closed symbols) monitored by measuring intrinsic tryptophan fluorescence. The inset shows the fluorescence curve of free
l
-tryptophan as a control. At each temperature, the fluorescence intensity relative to the signal at 25°C is indicated. The fluorescence curves are representative of three experiments.
FIG. 2
Temperature dependence of fluorescence change in a pyrene excimer fusion assay. Virions whose membranes had been metabolically labeled with pyrene-conjugated lipids were incubated with liposomes at various temperatures either at pH 5.5 or at pH 8.0, and the change in pyrene excimer fluorescence was monitored continuously. Black curves represent the pyrene-labeled virions incubated with liposomes at the indicated pH. Gray curves show the rate of fluorescence loss with pyrene-labeled virions that were incubated at pH 8.0 without liposomes as background controls. The data shown are representative of three experiments.
FIG. 3
Effects of elevated temperature on the oligomeric state of native and low-pH-treated E proteins. Virions that had been pretreated at pH 8.0 (solid line and open symbols) or pH 5.5 (dashed line and closed symbols) were incubated for 10 min either at 37°C (A) or at 50°C (B), solubilized with Triton X-100, and analyzed by sedimentation in 7 to 20% sucrose gradients containing 0.1% Triton X-100. The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), trimer (T), and the pellet (P) are indicated. The data shown are representative of five experiments.
FIG. 4
Temperature dependence of aggregation. Virions that had been pretreated at pH 8.0 (A) or pH 5.5 (B) were incubated at various temperatures, solubilized with Triton X-100, and analyzed by sedimentation in sucrose gradients as described in the legend for Fig. 3. The symbols indicate the percentages of soluble (open circles) or pelleted (closed circles) E protein. Each curve is the average of at least three experiments. Arrows indicate the samples that were used for the sedimentation analysis shown in Fig. 5.
FIG. 5
Oligomeric state of the E protein of the samples indicated by arrows in Fig. 4. Virions that had been pretreated at pH 8.0 (solid line and open symbols) or pH 5.5 (dashed line and closed symbols) were incubated for 10 min at either 45°C (solid line and open symbols) or 55°C (dashed line and closed symbols), solubilized with Triton X-100, and analyzed by sedimentation in 7 to 20% sucrose gradients containing 0.1% Triton X-100. The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), trimer (T), and the pellet (P) are indicated. The data shown are representative of three experiments.
FIG. 6
Schematic model of low-pH- and heat-induced structural alterations of the E protein of TBE virus. Upon exposure to low pH, native E dimers dissociate reversibly into monomers, which are subsequently converted in an irreversible step into homotrimers. Heating alone does not induce this transition but instead causes the E dimers to denature and aggregate. In contrast, E trimers obtained after incubation at an acidic pH are more thermostable and retain their oligomeric state at 50°C. We propose that protonation of the native E dimer, caused by exposure to low pH, is essential for shifting the equilibrium toward the monomeric form (Monomer+), thereby providing the intermediate required for the trimerization step. The question mark indicates that it is still not known whether a hypothetical unprotonated monomer (Monomer0) would be able to trimerize at a neutral pH.
References
- Baker D, Agard D A. Influenza hemagglutinin: kinetic control of protein function. Structure. 1994;2:907–910. - PubMed
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