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Identification of specific histidines as pH sensors in flavivirus membrane fusion.

Fritz R, Stiasny K, Heinz FX - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Medical University of Vienna, 1095 Vienna, Austria.

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.

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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.
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fig3: 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.

Mentions: It was the primary hypothesis of our work that histidines play a role as pH sensors and that their protonation would be the initial trigger for fusion. To analyze the very first step of the fusion process, we made use of an FP-specific mAb (A1) and developed an assay that allowed us to measure the acidic pH–induced disengagement of the FP from its protecting interactions in the E dimer and its exposure to the environment. In previous experiments, we had found that during conversion of E from the prefusion dimer to the postfusion trimer the accessibility of the FP for mAb A1 was transiently increased but lost upon conversion into the final trimeric conformation (Stiasny et al., 2007). We therefore measured this transient FP exposure using an enzyme immunoassay in which the antibody was already present in the RSP samples at the time of their acidification (see Materials and methods). The results of these experiments are shown in Fig. 3. With the exception of mutant H323A, none of the three other single His mutants differed significantly from the WT, neither with respect to the extent nor to the pH threshold of FP exposure (unpublished data). The same holds true for the double and triple mutants, which all displayed a WT-like pattern (Fig. 3). In contrast, FP exposure was severely impaired in the case of the H323A mutant, but the residual activity was induced at the same pH threshold as with the WT, i.e., around pH 6.6 (Fig. 3). These results allow us to conclude that of the four histidines analyzed only His323 plays an important role as an initial fusion trigger and that the lack of fusion activity observed with the double mutant H248N-H287A was caused by an impairment of later steps of fusion.


Identification of specific histidines as pH sensors in flavivirus membrane fusion.

Fritz R, Stiasny K, Heinz FX - J. Cell Biol. (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568029&req=5

fig3: 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.
Mentions: It was the primary hypothesis of our work that histidines play a role as pH sensors and that their protonation would be the initial trigger for fusion. To analyze the very first step of the fusion process, we made use of an FP-specific mAb (A1) and developed an assay that allowed us to measure the acidic pH–induced disengagement of the FP from its protecting interactions in the E dimer and its exposure to the environment. In previous experiments, we had found that during conversion of E from the prefusion dimer to the postfusion trimer the accessibility of the FP for mAb A1 was transiently increased but lost upon conversion into the final trimeric conformation (Stiasny et al., 2007). We therefore measured this transient FP exposure using an enzyme immunoassay in which the antibody was already present in the RSP samples at the time of their acidification (see Materials and methods). The results of these experiments are shown in Fig. 3. With the exception of mutant H323A, none of the three other single His mutants differed significantly from the WT, neither with respect to the extent nor to the pH threshold of FP exposure (unpublished data). The same holds true for the double and triple mutants, which all displayed a WT-like pattern (Fig. 3). In contrast, FP exposure was severely impaired in the case of the H323A mutant, but the residual activity was induced at the same pH threshold as with the WT, i.e., around pH 6.6 (Fig. 3). These results allow us to conclude that of the four histidines analyzed only His323 plays an important role as an initial fusion trigger and that the lack of fusion activity observed with the double mutant H248N-H287A was caused by an impairment of later steps of fusion.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Medical University of Vienna, 1095 Vienna, Austria.

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.

Show MeSH
Related in: MedlinePlus