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Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion.

Liao M, Kielian M - J. Cell Biol. (2005)

Bottom Line: During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides.Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion.These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Alphaviruses and flaviviruses infect cells through low pH-dependent membrane fusion reactions mediated by their structurally similar viral fusion proteins. During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides. We demonstrate that exogenous domain III can function as a dominant-negative inhibitor of alphavirus and flavivirus membrane fusion and infection. Domain III binds stably to the fusion protein, thus preventing the foldback reaction and blocking the lipid mixing step of fusion. Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion. These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

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SFV E1 domain III proteins inhibit the lipid mixing step of fusion. (A) Fluorescence scan of pyrene-labeled SFV fused with BHK cells. Pyrene-labeled SFV was prebound to BHK cells and incubated at 37°C for 1 min in pH 7.4 medium without domain III protein (curve a), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5 medium with 1 (curve c), 5 (curve d), or 8 μM (curve e) His-DIIIS. Background fluorescence from cells alone was subtracted and the fluorescence emission was normalized for each sample by setting the monomer peak at 397 nm to 5 (arbitrary units). Representative example of three experiments. (B) Comparison of inhibition of lipid mixing by domain III proteins. The fusion between pyrene-labeled SFV and BHK cells was assayed as in A, in the presence of the indicated concentrations of domain III proteins. The difference between the Ex/M at pH 7.4 and after treatment at pH 5.5 without domain III proteins was defined as 100% (control). The difference between the ratios of the pH 7.4 sample and each experimental sample was determined and expressed as a percentage of this control difference. Error bars are the mean ± SD. n = 3.
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fig5: SFV E1 domain III proteins inhibit the lipid mixing step of fusion. (A) Fluorescence scan of pyrene-labeled SFV fused with BHK cells. Pyrene-labeled SFV was prebound to BHK cells and incubated at 37°C for 1 min in pH 7.4 medium without domain III protein (curve a), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5 medium with 1 (curve c), 5 (curve d), or 8 μM (curve e) His-DIIIS. Background fluorescence from cells alone was subtracted and the fluorescence emission was normalized for each sample by setting the monomer peak at 397 nm to 5 (arbitrary units). Representative example of three experiments. (B) Comparison of inhibition of lipid mixing by domain III proteins. The fusion between pyrene-labeled SFV and BHK cells was assayed as in A, in the presence of the indicated concentrations of domain III proteins. The difference between the Ex/M at pH 7.4 and after treatment at pH 5.5 without domain III proteins was defined as 100% (control). The difference between the ratios of the pH 7.4 sample and each experimental sample was determined and expressed as a percentage of this control difference. Error bars are the mean ± SD. n = 3.

Mentions: Class II virus fusion initiates through the interaction of the fusion loop with the target membrane and progresses through an initial lipid mixing stage termed hemifusion in which the outer leaflets of the virus and target membranes mix (Zaitseva et al., 2005). This stage is followed by the opening of a fusion pore, which widens to give complete fusion and content mixing, the end stage of fusion monitored by the FIA. To test for the effects of domain III proteins on initial lipid mixing and hemifusion, we followed the loss of the pyrene excimer peak upon fusion of pyrene-labeled SFV with unlabeled target cells (Chatterjee et al., 2002). Pyrene-labeled SFV was bound to cells in the cold and pulsed at low pH in the presence or absence of exogenous domain III. We determined the fluorescence emission spectrum of each virus cell mixture and compared the excimer to monomer peak ratio (Ex/M). Untreated virus (unpublished data) or virus treated at pH 7.4 showed a strong excimer peak, with an Ex/M of ∼0.28 (Fig. 5 A, curve a). Virus treated at pH 5.5 showed efficient fusion with the cell plasma membrane, as reflected in the decrease of the excimer peak and an Ex/M of ∼0.10 (Fig. 5 A, curve b). The presence of His-DIIIS caused a concentration-dependent inhibition of the lipid mixing step (Fig. 5 A, curves c–e). No effect was observed when His-DIIIS was added to the sample after low pH treatment (unpublished data). As observed in the FIA, His-DIIIS showed the highest activity, with ∼90% inhibition of fusion at 8 μM (Fig. 5 B). Both His-DIII and DIIIS produced significant inhibition at 20 μM, whereas the DV domain III protein gave no inhibition at 20 μM. A higher concentration of His-DIIIS was required to completely inhibit pyrene virus fusion compared with the FIA, which could reflect an intrinsic difference in the inhibitor sensitivity of lipid mixing versus content mixing or the higher concentration of virus used in the pyrene versus FIA experiments.


Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion.

Liao M, Kielian M - J. Cell Biol. (2005)

SFV E1 domain III proteins inhibit the lipid mixing step of fusion. (A) Fluorescence scan of pyrene-labeled SFV fused with BHK cells. Pyrene-labeled SFV was prebound to BHK cells and incubated at 37°C for 1 min in pH 7.4 medium without domain III protein (curve a), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5 medium with 1 (curve c), 5 (curve d), or 8 μM (curve e) His-DIIIS. Background fluorescence from cells alone was subtracted and the fluorescence emission was normalized for each sample by setting the monomer peak at 397 nm to 5 (arbitrary units). Representative example of three experiments. (B) Comparison of inhibition of lipid mixing by domain III proteins. The fusion between pyrene-labeled SFV and BHK cells was assayed as in A, in the presence of the indicated concentrations of domain III proteins. The difference between the Ex/M at pH 7.4 and after treatment at pH 5.5 without domain III proteins was defined as 100% (control). The difference between the ratios of the pH 7.4 sample and each experimental sample was determined and expressed as a percentage of this control difference. Error bars are the mean ± SD. n = 3.
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fig5: SFV E1 domain III proteins inhibit the lipid mixing step of fusion. (A) Fluorescence scan of pyrene-labeled SFV fused with BHK cells. Pyrene-labeled SFV was prebound to BHK cells and incubated at 37°C for 1 min in pH 7.4 medium without domain III protein (curve a), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5 medium with 1 (curve c), 5 (curve d), or 8 μM (curve e) His-DIIIS. Background fluorescence from cells alone was subtracted and the fluorescence emission was normalized for each sample by setting the monomer peak at 397 nm to 5 (arbitrary units). Representative example of three experiments. (B) Comparison of inhibition of lipid mixing by domain III proteins. The fusion between pyrene-labeled SFV and BHK cells was assayed as in A, in the presence of the indicated concentrations of domain III proteins. The difference between the Ex/M at pH 7.4 and after treatment at pH 5.5 without domain III proteins was defined as 100% (control). The difference between the ratios of the pH 7.4 sample and each experimental sample was determined and expressed as a percentage of this control difference. Error bars are the mean ± SD. n = 3.
Mentions: Class II virus fusion initiates through the interaction of the fusion loop with the target membrane and progresses through an initial lipid mixing stage termed hemifusion in which the outer leaflets of the virus and target membranes mix (Zaitseva et al., 2005). This stage is followed by the opening of a fusion pore, which widens to give complete fusion and content mixing, the end stage of fusion monitored by the FIA. To test for the effects of domain III proteins on initial lipid mixing and hemifusion, we followed the loss of the pyrene excimer peak upon fusion of pyrene-labeled SFV with unlabeled target cells (Chatterjee et al., 2002). Pyrene-labeled SFV was bound to cells in the cold and pulsed at low pH in the presence or absence of exogenous domain III. We determined the fluorescence emission spectrum of each virus cell mixture and compared the excimer to monomer peak ratio (Ex/M). Untreated virus (unpublished data) or virus treated at pH 7.4 showed a strong excimer peak, with an Ex/M of ∼0.28 (Fig. 5 A, curve a). Virus treated at pH 5.5 showed efficient fusion with the cell plasma membrane, as reflected in the decrease of the excimer peak and an Ex/M of ∼0.10 (Fig. 5 A, curve b). The presence of His-DIIIS caused a concentration-dependent inhibition of the lipid mixing step (Fig. 5 A, curves c–e). No effect was observed when His-DIIIS was added to the sample after low pH treatment (unpublished data). As observed in the FIA, His-DIIIS showed the highest activity, with ∼90% inhibition of fusion at 8 μM (Fig. 5 B). Both His-DIII and DIIIS produced significant inhibition at 20 μM, whereas the DV domain III protein gave no inhibition at 20 μM. A higher concentration of His-DIIIS was required to completely inhibit pyrene virus fusion compared with the FIA, which could reflect an intrinsic difference in the inhibitor sensitivity of lipid mixing versus content mixing or the higher concentration of virus used in the pyrene versus FIA experiments.

Bottom Line: During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides.Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion.These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Alphaviruses and flaviviruses infect cells through low pH-dependent membrane fusion reactions mediated by their structurally similar viral fusion proteins. During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides. We demonstrate that exogenous domain III can function as a dominant-negative inhibitor of alphavirus and flavivirus membrane fusion and infection. Domain III binds stably to the fusion protein, thus preventing the foldback reaction and blocking the lipid mixing step of fusion. Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion. These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III-core trimer interaction can serve as a new target for the development of antiviral reagents.

Show MeSH
Related in: MedlinePlus