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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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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.
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fig6: 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.

Mentions: The results obtained so far indicated that only His323, but none of the other histidines investigated, was involved in the initiation of fusion. We therefore hypothesized that the lack of fusion observed with the double mutant H248N-H287A was caused by an impairment of later steps, such as the formation and/or stability of the E trimer. We addressed this question by sedimentation analyses and investigated the effect of each of the His mutations on the acidic pH–induced conversion of E dimers into trimers. Like with the WT RSPs, a quantitative E dimer to trimer transition was observed with the single mutants H248N, H287A, and H438N, as well as with the double mutants H248N-H438N and H287A-H438N (Fig. 5, A and B; and not depicted). In contrast, trimer formation was impaired with H323A and H248N-H287A (Fig. 5, C and D). Similar to the situation found with the pH dependence of FP exposure (Fig. 3), the pH threshold for the oligomeric rearrangement was identical to the WT with all of the mutants (pH 6.6), irrespective of the amount of trimers formed (Fig. 6).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: 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.
Mentions: The results obtained so far indicated that only His323, but none of the other histidines investigated, was involved in the initiation of fusion. We therefore hypothesized that the lack of fusion observed with the double mutant H248N-H287A was caused by an impairment of later steps, such as the formation and/or stability of the E trimer. We addressed this question by sedimentation analyses and investigated the effect of each of the His mutations on the acidic pH–induced conversion of E dimers into trimers. Like with the WT RSPs, a quantitative E dimer to trimer transition was observed with the single mutants H248N, H287A, and H438N, as well as with the double mutants H248N-H438N and H287A-H438N (Fig. 5, A and B; and not depicted). In contrast, trimer formation was impaired with H323A and H248N-H287A (Fig. 5, C and D). Similar to the situation found with the pH dependence of FP exposure (Fig. 3), the pH threshold for the oligomeric rearrangement was identical to the WT with all of the mutants (pH 6.6), irrespective of the amount of trimers formed (Fig. 6).

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