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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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Summary of domain III proteins. (A) Structure of the SFV E1 ectodomain in the neutral pH monomer conformation (left; modified from Gibbons et al., 2004b) and in the low pH-induced trimer conformation (right), showing a single E1 protein of the trimer (drawn using PyMOL; DeLano, 2002). The colors indicate domains I (red), II (yellow), and III (blue), and the fusion loop (fl; orange) at the tip of domain II. The movement of domain III and the stem toward the fusion loop is indicated by the small black arrow. (B) Linear diagram of sequences of SFV E1 and DV2 E and domain III constructs, showing the boundaries of the domains, stem region, and TM anchor region. The SFV E1 domain III proteins are as follows: DIII (residues 291–383), DIIIS (291–412), His-DIII (His tag plus 291–383), and His-DIIIS (His tag plus 291–412). The DV2 E domain III proteins are as follows: DV2DIIIH1 (296–415) and His-DV2DIII (His tag plus 296–395). The His tag adds 36 residues at the NH2 terminus; untagged proteins contain an added methionine at the NH2 terminus. (C) 2 μg of each purified domain III protein was treated with or without 10 mM DTT, alkylated, and analyzed by SDS-PAGE. Marker proteins are shown on the left with their molecular masses listed in kilodaltons. (D) The molecular mass of each domain III protein was measured by mass spectrometry and compared with the mass calculated from the amino acid sequence. The predicted error rate is 0.01%. The mass for DV2DIIIH1was calculated without the added NH2-terminal methionine because the measured mass indicated that this residue was not contained in the protein. (E) Elution profiles of 50 μM His-DIIIS and DIIIS on Superdex G-75 in 0.1 M Na Acetate, pH 5.5.
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fig1: Summary of domain III proteins. (A) Structure of the SFV E1 ectodomain in the neutral pH monomer conformation (left; modified from Gibbons et al., 2004b) and in the low pH-induced trimer conformation (right), showing a single E1 protein of the trimer (drawn using PyMOL; DeLano, 2002). The colors indicate domains I (red), II (yellow), and III (blue), and the fusion loop (fl; orange) at the tip of domain II. The movement of domain III and the stem toward the fusion loop is indicated by the small black arrow. (B) Linear diagram of sequences of SFV E1 and DV2 E and domain III constructs, showing the boundaries of the domains, stem region, and TM anchor region. The SFV E1 domain III proteins are as follows: DIII (residues 291–383), DIIIS (291–412), His-DIII (His tag plus 291–383), and His-DIIIS (His tag plus 291–412). The DV2 E domain III proteins are as follows: DV2DIIIH1 (296–415) and His-DV2DIII (His tag plus 296–395). The His tag adds 36 residues at the NH2 terminus; untagged proteins contain an added methionine at the NH2 terminus. (C) 2 μg of each purified domain III protein was treated with or without 10 mM DTT, alkylated, and analyzed by SDS-PAGE. Marker proteins are shown on the left with their molecular masses listed in kilodaltons. (D) The molecular mass of each domain III protein was measured by mass spectrometry and compared with the mass calculated from the amino acid sequence. The predicted error rate is 0.01%. The mass for DV2DIIIH1was calculated without the added NH2-terminal methionine because the measured mass indicated that this residue was not contained in the protein. (E) Elution profiles of 50 μM His-DIIIS and DIIIS on Superdex G-75 in 0.1 M Na Acetate, pH 5.5.

Mentions: Although the alphavirus and flavivirus fusion proteins do not have detectable amino acid sequence similarity, they have remarkably similar secondary and tertiary structures, indicating their evolutionary relationship and leading to their classification as the inaugural members of the class II virus fusion proteins (Lescar et al., 2001). The neutral pH structures of the fusion protein ectodomains have been determined for the alphavirus Semliki Forest virus (SFV; Lescar et al., 2001) and the flaviviruses TBE, DV2, and DV3 (Rey et al., 1995; Modis et al., 2003, 2005; Zhang et al., 2004). The proteins are elongated molecules composed almost entirely of β strands and contain three domains: the centrally located domain I; domain II, which is located at one side of domain I and contains the target-membrane–interacting fusion peptide loop at its tip; and an Ig-like domain III, which is connected to the other side of domain I (Fig. 1 A). Although not present in the ectodomain structure, in the full-length proteins the stem region and TM anchor are found at the COOH terminus of domain III, at the opposite end of the protein from the fusion loop. The fusion proteins are arranged with icosahedral symmetry and lie tangential (almost parallel) to the virus membrane (Lescar et al., 2001; Kuhn et al., 2002; W. Zhang et al., 2002).


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)

Summary of domain III proteins. (A) Structure of the SFV E1 ectodomain in the neutral pH monomer conformation (left; modified from Gibbons et al., 2004b) and in the low pH-induced trimer conformation (right), showing a single E1 protein of the trimer (drawn using PyMOL; DeLano, 2002). The colors indicate domains I (red), II (yellow), and III (blue), and the fusion loop (fl; orange) at the tip of domain II. The movement of domain III and the stem toward the fusion loop is indicated by the small black arrow. (B) Linear diagram of sequences of SFV E1 and DV2 E and domain III constructs, showing the boundaries of the domains, stem region, and TM anchor region. The SFV E1 domain III proteins are as follows: DIII (residues 291–383), DIIIS (291–412), His-DIII (His tag plus 291–383), and His-DIIIS (His tag plus 291–412). The DV2 E domain III proteins are as follows: DV2DIIIH1 (296–415) and His-DV2DIII (His tag plus 296–395). The His tag adds 36 residues at the NH2 terminus; untagged proteins contain an added methionine at the NH2 terminus. (C) 2 μg of each purified domain III protein was treated with or without 10 mM DTT, alkylated, and analyzed by SDS-PAGE. Marker proteins are shown on the left with their molecular masses listed in kilodaltons. (D) The molecular mass of each domain III protein was measured by mass spectrometry and compared with the mass calculated from the amino acid sequence. The predicted error rate is 0.01%. The mass for DV2DIIIH1was calculated without the added NH2-terminal methionine because the measured mass indicated that this residue was not contained in the protein. (E) Elution profiles of 50 μM His-DIIIS and DIIIS on Superdex G-75 in 0.1 M Na Acetate, pH 5.5.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171229&req=5

fig1: Summary of domain III proteins. (A) Structure of the SFV E1 ectodomain in the neutral pH monomer conformation (left; modified from Gibbons et al., 2004b) and in the low pH-induced trimer conformation (right), showing a single E1 protein of the trimer (drawn using PyMOL; DeLano, 2002). The colors indicate domains I (red), II (yellow), and III (blue), and the fusion loop (fl; orange) at the tip of domain II. The movement of domain III and the stem toward the fusion loop is indicated by the small black arrow. (B) Linear diagram of sequences of SFV E1 and DV2 E and domain III constructs, showing the boundaries of the domains, stem region, and TM anchor region. The SFV E1 domain III proteins are as follows: DIII (residues 291–383), DIIIS (291–412), His-DIII (His tag plus 291–383), and His-DIIIS (His tag plus 291–412). The DV2 E domain III proteins are as follows: DV2DIIIH1 (296–415) and His-DV2DIII (His tag plus 296–395). The His tag adds 36 residues at the NH2 terminus; untagged proteins contain an added methionine at the NH2 terminus. (C) 2 μg of each purified domain III protein was treated with or without 10 mM DTT, alkylated, and analyzed by SDS-PAGE. Marker proteins are shown on the left with their molecular masses listed in kilodaltons. (D) The molecular mass of each domain III protein was measured by mass spectrometry and compared with the mass calculated from the amino acid sequence. The predicted error rate is 0.01%. The mass for DV2DIIIH1was calculated without the added NH2-terminal methionine because the measured mass indicated that this residue was not contained in the protein. (E) Elution profiles of 50 μM His-DIIIS and DIIIS on Superdex G-75 in 0.1 M Na Acetate, pH 5.5.
Mentions: Although the alphavirus and flavivirus fusion proteins do not have detectable amino acid sequence similarity, they have remarkably similar secondary and tertiary structures, indicating their evolutionary relationship and leading to their classification as the inaugural members of the class II virus fusion proteins (Lescar et al., 2001). The neutral pH structures of the fusion protein ectodomains have been determined for the alphavirus Semliki Forest virus (SFV; Lescar et al., 2001) and the flaviviruses TBE, DV2, and DV3 (Rey et al., 1995; Modis et al., 2003, 2005; Zhang et al., 2004). The proteins are elongated molecules composed almost entirely of β strands and contain three domains: the centrally located domain I; domain II, which is located at one side of domain I and contains the target-membrane–interacting fusion peptide loop at its tip; and an Ig-like domain III, which is connected to the other side of domain I (Fig. 1 A). Although not present in the ectodomain structure, in the full-length proteins the stem region and TM anchor are found at the COOH terminus of domain III, at the opposite end of the protein from the fusion loop. The fusion proteins are arranged with icosahedral symmetry and lie tangential (almost parallel) to the virus membrane (Lescar et al., 2001; Kuhn et al., 2002; W. Zhang et al., 2002).

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