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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: 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.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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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.
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fig2: 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.

Mentions: Having certified that the replacement of histidines by other amino acids had not changed the overall conformation of E and its oligomeric state (nor did it impair particle formation, maturation, or secretion), we analyzed the effect of these mutations on fusion activity. For this purpose, the membranes of each of the mutant RSPs were fluorescence labeled in vivo with 1-pyrenehexadecanoic acid and subjected to an in vitro fusion assay (see Materials and methods). These RSPs were mixed with unlabeled liposomes and the decrease in pyrene excimer fluorescence (caused by dilution of the probe in the target membrane) was continuously monitored. Consistent with previous results (Corver et al., 2000), WT RSPs fused rapidly at pH 5.4 within the first seconds after acidification (Fig. 2 A), and the fusion activities of mutants lacking a histidine residue at position 248, 287, or 438 were identical to that of the WT (Fig. 2 B). The replacement of His323, however, resulted in the loss of fusion activity (Fig. 2, A and B), even at pH 5.0 (not depicted). We also analyzed combinations of mutations that alone did not affect fusion, i.e., H248N+H287A, H248N+H438N, and H287A+H438N. Unexpectedly, one of these combinations (H248N+H287A) impaired fusion to the same extent as did the replacement of His323 (Fig. 2, A and B). These experiments revealed the importance of individual conserved histidines for E-mediated fusion, but it remained unclear at which stage or how certain residues were involved in this multistep process.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: Having certified that the replacement of histidines by other amino acids had not changed the overall conformation of E and its oligomeric state (nor did it impair particle formation, maturation, or secretion), we analyzed the effect of these mutations on fusion activity. For this purpose, the membranes of each of the mutant RSPs were fluorescence labeled in vivo with 1-pyrenehexadecanoic acid and subjected to an in vitro fusion assay (see Materials and methods). These RSPs were mixed with unlabeled liposomes and the decrease in pyrene excimer fluorescence (caused by dilution of the probe in the target membrane) was continuously monitored. Consistent with previous results (Corver et al., 2000), WT RSPs fused rapidly at pH 5.4 within the first seconds after acidification (Fig. 2 A), and the fusion activities of mutants lacking a histidine residue at position 248, 287, or 438 were identical to that of the WT (Fig. 2 B). The replacement of His323, however, resulted in the loss of fusion activity (Fig. 2, A and B), even at pH 5.0 (not depicted). We also analyzed combinations of mutations that alone did not affect fusion, i.e., H248N+H287A, H248N+H438N, and H287A+H438N. Unexpectedly, one of these combinations (H248N+H287A) impaired fusion to the same extent as did the replacement of His323 (Fig. 2, A and B). These experiments revealed the importance of individual conserved histidines for E-mediated fusion, but it remained unclear at which stage or how certain residues were involved in this multistep process.

Bottom Line: 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.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