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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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Model for SFV E1 conformational changes during fusion and the action of exogenous domain III. (A) Prefusion form of E1 on the virus surface, with the E2 protein shown in gray, the E1 domains colored as in Fig. 1 A, and the fusion loop indicated by an orange star. The virus membrane is shown in brown and the target membrane is shown in blue. At this stage E1 is mAb E1a-1negative, trypsin sensitive, and shows no SDS-resistant trimer band (Kielian et al., 2000). (B) Low pH triggers the dissociation of E2/E1 dimer and the initial interaction of monomeric E1 with the target membrane. (C) Proposed membrane-inserted E1 trimer, suggested as a relatively long-lived intermediate. Subsequent folding back of domain III and the stem region would drive membrane fusion. (D) Postfusion HT form of E1 with domain III and the stem region (gray) fully folded back. In this conformation, the E1 trimer is mAb E1a-1 positive, trypsin resistant, and SDS resistant (Kielian et al., 2000). (C′/C′′) This panel illustrates the interaction of exogenous domain III (turquoise circle) with the proposed E1 trimer intermediate shown in C. This interaction produces a mixed population of domain III–bound trimers as illustrated by the two states, C′ and C′′, and the dotted line connecting them. All the states in the mixed population would be blocked from fusing and would differ in their conformation and biochemical properties. In the C′ state, with low concentration and/or low affinity of domain III proteins, some E1 trimers would undergo partial foldback and binding of one exogenous domain III. In the C′′ state, with high concentration and/or high affinity of domain III proteins, trimers would bind three exogenous domain III proteins and would be completely blocked in foldback. We predict that the C′ state would be mostly SDS resistant and mAb E1a-1 positive, whereas the C′′ state would be SDS sensitive and E1a-1 negative. This model is simplified and does not illustrate the stages of membrane curvature, the potential roles of cooperative trimer interactions, or the initial lipid mixing and pore formation.
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fig7: Model for SFV E1 conformational changes during fusion and the action of exogenous domain III. (A) Prefusion form of E1 on the virus surface, with the E2 protein shown in gray, the E1 domains colored as in Fig. 1 A, and the fusion loop indicated by an orange star. The virus membrane is shown in brown and the target membrane is shown in blue. At this stage E1 is mAb E1a-1negative, trypsin sensitive, and shows no SDS-resistant trimer band (Kielian et al., 2000). (B) Low pH triggers the dissociation of E2/E1 dimer and the initial interaction of monomeric E1 with the target membrane. (C) Proposed membrane-inserted E1 trimer, suggested as a relatively long-lived intermediate. Subsequent folding back of domain III and the stem region would drive membrane fusion. (D) Postfusion HT form of E1 with domain III and the stem region (gray) fully folded back. In this conformation, the E1 trimer is mAb E1a-1 positive, trypsin resistant, and SDS resistant (Kielian et al., 2000). (C′/C′′) This panel illustrates the interaction of exogenous domain III (turquoise circle) with the proposed E1 trimer intermediate shown in C. This interaction produces a mixed population of domain III–bound trimers as illustrated by the two states, C′ and C′′, and the dotted line connecting them. All the states in the mixed population would be blocked from fusing and would differ in their conformation and biochemical properties. In the C′ state, with low concentration and/or low affinity of domain III proteins, some E1 trimers would undergo partial foldback and binding of one exogenous domain III. In the C′′ state, with high concentration and/or high affinity of domain III proteins, trimers would bind three exogenous domain III proteins and would be completely blocked in foldback. We predict that the C′ state would be mostly SDS resistant and mAb E1a-1 positive, whereas the C′′ state would be SDS sensitive and E1a-1 negative. This model is simplified and does not illustrate the stages of membrane curvature, the potential roles of cooperative trimer interactions, or the initial lipid mixing and pore formation.

Mentions: Dominant-negative binding of exogenous domain III would be predicted to alter the conformation of the E1 HT by preventing the folding back of the viral domain III, and consequently could decrease trimer stability. Exposure of the acid-conformation–specific mAb E1a-1 epitope on domain I closely correlates with HT formation, although the epitope is not formed by trimerization per se (Ahn et al., 1999). Interestingly, concentrations of His-DIIIS above 2 μM led to a gradual decrease in the retrieval of E1 by both the anti-His antibody and mAb E1a-1 (Fig. 6 B). This suggests that the binding of exogenous domain III is directly affecting the conformation of the E1HT. Destabilization of the trimer structure by domain III could also explain why somewhat less trypsin-resistant trimer was recovered after retrieval with domain III, as compared with the acid-specific mAb (Fig. 6 C). We directly evaluated HT stability by following the resistance of the SFV HT to dissociation by SDS sample buffer at 30°C (Fig. 6 D). Increasing amounts of domain III proteins lead to the loss of the SDS-resistant HT conformation, with only 10% of the control HT observed in the presence of 10 μM His-DIIIS. Interestingly, bands migrating above and below the position of the E1HT were clearly observed with His-DIIIS, suggesting the presence of alternative E1 complexes. A decrease in the SDS-resistant E1HT was also observed in the presence of increasing amounts of His-DIII (60% of control HT at 10 μM His-DIII). Together, these results support a model (Fig. 7) in which exogenous domain III binds to an intermediate trimeric conformation of E1 and prevents final hairpin formation and fusion.


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)

Model for SFV E1 conformational changes during fusion and the action of exogenous domain III. (A) Prefusion form of E1 on the virus surface, with the E2 protein shown in gray, the E1 domains colored as in Fig. 1 A, and the fusion loop indicated by an orange star. The virus membrane is shown in brown and the target membrane is shown in blue. At this stage E1 is mAb E1a-1negative, trypsin sensitive, and shows no SDS-resistant trimer band (Kielian et al., 2000). (B) Low pH triggers the dissociation of E2/E1 dimer and the initial interaction of monomeric E1 with the target membrane. (C) Proposed membrane-inserted E1 trimer, suggested as a relatively long-lived intermediate. Subsequent folding back of domain III and the stem region would drive membrane fusion. (D) Postfusion HT form of E1 with domain III and the stem region (gray) fully folded back. In this conformation, the E1 trimer is mAb E1a-1 positive, trypsin resistant, and SDS resistant (Kielian et al., 2000). (C′/C′′) This panel illustrates the interaction of exogenous domain III (turquoise circle) with the proposed E1 trimer intermediate shown in C. This interaction produces a mixed population of domain III–bound trimers as illustrated by the two states, C′ and C′′, and the dotted line connecting them. All the states in the mixed population would be blocked from fusing and would differ in their conformation and biochemical properties. In the C′ state, with low concentration and/or low affinity of domain III proteins, some E1 trimers would undergo partial foldback and binding of one exogenous domain III. In the C′′ state, with high concentration and/or high affinity of domain III proteins, trimers would bind three exogenous domain III proteins and would be completely blocked in foldback. We predict that the C′ state would be mostly SDS resistant and mAb E1a-1 positive, whereas the C′′ state would be SDS sensitive and E1a-1 negative. This model is simplified and does not illustrate the stages of membrane curvature, the potential roles of cooperative trimer interactions, or the initial lipid mixing and pore formation.
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Related In: Results  -  Collection

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fig7: Model for SFV E1 conformational changes during fusion and the action of exogenous domain III. (A) Prefusion form of E1 on the virus surface, with the E2 protein shown in gray, the E1 domains colored as in Fig. 1 A, and the fusion loop indicated by an orange star. The virus membrane is shown in brown and the target membrane is shown in blue. At this stage E1 is mAb E1a-1negative, trypsin sensitive, and shows no SDS-resistant trimer band (Kielian et al., 2000). (B) Low pH triggers the dissociation of E2/E1 dimer and the initial interaction of monomeric E1 with the target membrane. (C) Proposed membrane-inserted E1 trimer, suggested as a relatively long-lived intermediate. Subsequent folding back of domain III and the stem region would drive membrane fusion. (D) Postfusion HT form of E1 with domain III and the stem region (gray) fully folded back. In this conformation, the E1 trimer is mAb E1a-1 positive, trypsin resistant, and SDS resistant (Kielian et al., 2000). (C′/C′′) This panel illustrates the interaction of exogenous domain III (turquoise circle) with the proposed E1 trimer intermediate shown in C. This interaction produces a mixed population of domain III–bound trimers as illustrated by the two states, C′ and C′′, and the dotted line connecting them. All the states in the mixed population would be blocked from fusing and would differ in their conformation and biochemical properties. In the C′ state, with low concentration and/or low affinity of domain III proteins, some E1 trimers would undergo partial foldback and binding of one exogenous domain III. In the C′′ state, with high concentration and/or high affinity of domain III proteins, trimers would bind three exogenous domain III proteins and would be completely blocked in foldback. We predict that the C′ state would be mostly SDS resistant and mAb E1a-1 positive, whereas the C′′ state would be SDS sensitive and E1a-1 negative. This model is simplified and does not illustrate the stages of membrane curvature, the potential roles of cooperative trimer interactions, or the initial lipid mixing and pore formation.
Mentions: Dominant-negative binding of exogenous domain III would be predicted to alter the conformation of the E1 HT by preventing the folding back of the viral domain III, and consequently could decrease trimer stability. Exposure of the acid-conformation–specific mAb E1a-1 epitope on domain I closely correlates with HT formation, although the epitope is not formed by trimerization per se (Ahn et al., 1999). Interestingly, concentrations of His-DIIIS above 2 μM led to a gradual decrease in the retrieval of E1 by both the anti-His antibody and mAb E1a-1 (Fig. 6 B). This suggests that the binding of exogenous domain III is directly affecting the conformation of the E1HT. Destabilization of the trimer structure by domain III could also explain why somewhat less trypsin-resistant trimer was recovered after retrieval with domain III, as compared with the acid-specific mAb (Fig. 6 C). We directly evaluated HT stability by following the resistance of the SFV HT to dissociation by SDS sample buffer at 30°C (Fig. 6 D). Increasing amounts of domain III proteins lead to the loss of the SDS-resistant HT conformation, with only 10% of the control HT observed in the presence of 10 μM His-DIIIS. Interestingly, bands migrating above and below the position of the E1HT were clearly observed with His-DIIIS, suggesting the presence of alternative E1 complexes. A decrease in the SDS-resistant E1HT was also observed in the presence of increasing amounts of His-DIII (60% of control HT at 10 μM His-DIII). Together, these results support a model (Fig. 7) in which exogenous domain III binds to an intermediate trimeric conformation of E1 and prevents final hairpin formation and fusion.

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