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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 domain III proteins bind to trimeric E1 during the fusion reaction. (A) Domain III proteins bind to E1 during fusion. 35S-labeled SFV was bound to BHK cells on ice and treated at pH 7.4 or 5.5 at 37°C for 1 min in the presence of the indicated domain III proteins. Cells were washed, lysed, and immunoprecipitated with a rabbit polyclonal antibody against the SFV E1 and E2 protein (Rab), a mAb against the low pH conformation of E1 (E1a-1), a mAb against the His tag (HIS-1), a rabbit preimmune serum (Pre), or an isotype-matched irrelevant mAb (12G5). Samples were analyzed by SDS-PAGE and fluorography. (B) Quantitation of samples prepared as in A using the indicated concentrations of His-DIII or His-DIIIS. N indicates 1 min treatment at pH 7.4 with 2 μM His-DIIIS. The total E1 in each sample was defined as the amount of E1 immunoprecipitated by Rab. Representative example of two experiments. (C) Domain III selectively interacts with a trimeric form of E1. Fusion reactions were triggered at pH 7.4 or 5.5 in the presence of 10 μM His-DIII as in A. Samples were immunoprecipitated with the indicated antibodies, digested with trypsin where indicated, and analyzed by SDS-PAGE. The amount of trypsin-resistant E1 was quantitated and expressed as a percentage of the nontrypsinized E1 for each sample. Error bars are the mean ± SD. n = 3. (D) Exogenous domain III proteins affect the SDS-resistant conformation of the E1 HT. Samples were prepared as in B. An aliquot of the cell lysate was treated with SDS-sample buffer at 30°C and analyzed by SDS-PAGE and fluorography. For each sample, the SDS-resistant E1HT band (arrow) was quantitated and expressed as a percentage of the E1HT in the absence of domain III proteins. Representative example of two experiments.
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fig6: SFV domain III proteins bind to trimeric E1 during the fusion reaction. (A) Domain III proteins bind to E1 during fusion. 35S-labeled SFV was bound to BHK cells on ice and treated at pH 7.4 or 5.5 at 37°C for 1 min in the presence of the indicated domain III proteins. Cells were washed, lysed, and immunoprecipitated with a rabbit polyclonal antibody against the SFV E1 and E2 protein (Rab), a mAb against the low pH conformation of E1 (E1a-1), a mAb against the His tag (HIS-1), a rabbit preimmune serum (Pre), or an isotype-matched irrelevant mAb (12G5). Samples were analyzed by SDS-PAGE and fluorography. (B) Quantitation of samples prepared as in A using the indicated concentrations of His-DIII or His-DIIIS. N indicates 1 min treatment at pH 7.4 with 2 μM His-DIIIS. The total E1 in each sample was defined as the amount of E1 immunoprecipitated by Rab. Representative example of two experiments. (C) Domain III selectively interacts with a trimeric form of E1. Fusion reactions were triggered at pH 7.4 or 5.5 in the presence of 10 μM His-DIII as in A. Samples were immunoprecipitated with the indicated antibodies, digested with trypsin where indicated, and analyzed by SDS-PAGE. The amount of trypsin-resistant E1 was quantitated and expressed as a percentage of the nontrypsinized E1 for each sample. Error bars are the mean ± SD. n = 3. (D) Exogenous domain III proteins affect the SDS-resistant conformation of the E1 HT. Samples were prepared as in B. An aliquot of the cell lysate was treated with SDS-sample buffer at 30°C and analyzed by SDS-PAGE and fluorography. For each sample, the SDS-resistant E1HT band (arrow) was quantitated and expressed as a percentage of the E1HT in the absence of domain III proteins. Representative example of two experiments.

Mentions: We screened the SFV DIII proteins for activity in a fusion-infection assay (FIA) that quantitates low pH-dependent SFV fusion with the plasma membrane (Vashishtha et al., 1998). Viruses were bound to cells on ice and treated for 1 min at 37°C at low pH to trigger the fusion of the virus with the plasma membrane of the cell. This fusion results in virus infection. The cells were cultured overnight in the presence of 20 mM NH4Cl to prevent secondary infection, and the cells infected due to the low pH pulse were quantitated by immunofluorescence. Under these conditions, we could test the effects of domain III proteins specifically during the binding step, the fusion step, and the postfusion culture step. As shown in Fig. 2 A, 4 μM His-DIII almost completely inhibited SFV infection of BHK cells, but only when present during the low pH-induced fusion step. Similar results were obtained for His-DIIIS (unpublished data). In contrast, preincubation of the virus with domain III proteins at 37°C at neutral pH had no effect (unpublished data). In agreement with studies showing that alphavirus receptor interaction is mediated by the E2 protein (for review see Schlesinger and Schlesinger, 2001), exogenous domain III proteins did not inhibit virus cell binding or release prebound virus from cells (Fig. 2 A and see Fig. 6). Inhibition by domain III protein was comparable when virus was prebound to cells at pH 6.5, 6.8, 7.4, or 8.0, or when the low pH pulse was at pH 5.5 or 6.0 (unpublished data). Comparison of the four SFV domain III proteins showed that the strongest inhibition was obtained with His-DIIIS (IC50 ∼0.1 μM), followed by His-DIII (IC50 ∼0.5 μM), DIIIS (IC50 ∼6 μM), and DIII, which gave ∼40% inhibition at a concentration of 80 μM (Fig. 2 B). Thus, the presence of both the stem region and the NH2-terminal His tag resulted in increased effectiveness. Although enhancement by the stem region is suggested from the structure of the low pH-induced HT, the reason for the increase in inhibition observed with His-tagged forms of SFV domain III is not known. The tag at the domain III NH2 terminus could act by stabilizing binding to E1, mimicking the important domain I–domain III linker region and/or enhancing its trimeric interactions, concentrating the protein at the membrane at low pH, preventing displacement of the exogenous DIII by the endogenous DIII, and/or preventing cooperative HT–HT interactions. High concentrations of His-tagged DV2 domain III protein did not affect SFV fusion (Fig. 3 B), indicating that there is no nonspecific effect of the His tag.


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 domain III proteins bind to trimeric E1 during the fusion reaction. (A) Domain III proteins bind to E1 during fusion. 35S-labeled SFV was bound to BHK cells on ice and treated at pH 7.4 or 5.5 at 37°C for 1 min in the presence of the indicated domain III proteins. Cells were washed, lysed, and immunoprecipitated with a rabbit polyclonal antibody against the SFV E1 and E2 protein (Rab), a mAb against the low pH conformation of E1 (E1a-1), a mAb against the His tag (HIS-1), a rabbit preimmune serum (Pre), or an isotype-matched irrelevant mAb (12G5). Samples were analyzed by SDS-PAGE and fluorography. (B) Quantitation of samples prepared as in A using the indicated concentrations of His-DIII or His-DIIIS. N indicates 1 min treatment at pH 7.4 with 2 μM His-DIIIS. The total E1 in each sample was defined as the amount of E1 immunoprecipitated by Rab. Representative example of two experiments. (C) Domain III selectively interacts with a trimeric form of E1. Fusion reactions were triggered at pH 7.4 or 5.5 in the presence of 10 μM His-DIII as in A. Samples were immunoprecipitated with the indicated antibodies, digested with trypsin where indicated, and analyzed by SDS-PAGE. The amount of trypsin-resistant E1 was quantitated and expressed as a percentage of the nontrypsinized E1 for each sample. Error bars are the mean ± SD. n = 3. (D) Exogenous domain III proteins affect the SDS-resistant conformation of the E1 HT. Samples were prepared as in B. An aliquot of the cell lysate was treated with SDS-sample buffer at 30°C and analyzed by SDS-PAGE and fluorography. For each sample, the SDS-resistant E1HT band (arrow) was quantitated and expressed as a percentage of the E1HT in the absence of domain III proteins. Representative example of two experiments.
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fig6: SFV domain III proteins bind to trimeric E1 during the fusion reaction. (A) Domain III proteins bind to E1 during fusion. 35S-labeled SFV was bound to BHK cells on ice and treated at pH 7.4 or 5.5 at 37°C for 1 min in the presence of the indicated domain III proteins. Cells were washed, lysed, and immunoprecipitated with a rabbit polyclonal antibody against the SFV E1 and E2 protein (Rab), a mAb against the low pH conformation of E1 (E1a-1), a mAb against the His tag (HIS-1), a rabbit preimmune serum (Pre), or an isotype-matched irrelevant mAb (12G5). Samples were analyzed by SDS-PAGE and fluorography. (B) Quantitation of samples prepared as in A using the indicated concentrations of His-DIII or His-DIIIS. N indicates 1 min treatment at pH 7.4 with 2 μM His-DIIIS. The total E1 in each sample was defined as the amount of E1 immunoprecipitated by Rab. Representative example of two experiments. (C) Domain III selectively interacts with a trimeric form of E1. Fusion reactions were triggered at pH 7.4 or 5.5 in the presence of 10 μM His-DIII as in A. Samples were immunoprecipitated with the indicated antibodies, digested with trypsin where indicated, and analyzed by SDS-PAGE. The amount of trypsin-resistant E1 was quantitated and expressed as a percentage of the nontrypsinized E1 for each sample. Error bars are the mean ± SD. n = 3. (D) Exogenous domain III proteins affect the SDS-resistant conformation of the E1 HT. Samples were prepared as in B. An aliquot of the cell lysate was treated with SDS-sample buffer at 30°C and analyzed by SDS-PAGE and fluorography. For each sample, the SDS-resistant E1HT band (arrow) was quantitated and expressed as a percentage of the E1HT in the absence of domain III proteins. Representative example of two experiments.
Mentions: We screened the SFV DIII proteins for activity in a fusion-infection assay (FIA) that quantitates low pH-dependent SFV fusion with the plasma membrane (Vashishtha et al., 1998). Viruses were bound to cells on ice and treated for 1 min at 37°C at low pH to trigger the fusion of the virus with the plasma membrane of the cell. This fusion results in virus infection. The cells were cultured overnight in the presence of 20 mM NH4Cl to prevent secondary infection, and the cells infected due to the low pH pulse were quantitated by immunofluorescence. Under these conditions, we could test the effects of domain III proteins specifically during the binding step, the fusion step, and the postfusion culture step. As shown in Fig. 2 A, 4 μM His-DIII almost completely inhibited SFV infection of BHK cells, but only when present during the low pH-induced fusion step. Similar results were obtained for His-DIIIS (unpublished data). In contrast, preincubation of the virus with domain III proteins at 37°C at neutral pH had no effect (unpublished data). In agreement with studies showing that alphavirus receptor interaction is mediated by the E2 protein (for review see Schlesinger and Schlesinger, 2001), exogenous domain III proteins did not inhibit virus cell binding or release prebound virus from cells (Fig. 2 A and see Fig. 6). Inhibition by domain III protein was comparable when virus was prebound to cells at pH 6.5, 6.8, 7.4, or 8.0, or when the low pH pulse was at pH 5.5 or 6.0 (unpublished data). Comparison of the four SFV domain III proteins showed that the strongest inhibition was obtained with His-DIIIS (IC50 ∼0.1 μM), followed by His-DIII (IC50 ∼0.5 μM), DIIIS (IC50 ∼6 μM), and DIII, which gave ∼40% inhibition at a concentration of 80 μM (Fig. 2 B). Thus, the presence of both the stem region and the NH2-terminal His tag resulted in increased effectiveness. Although enhancement by the stem region is suggested from the structure of the low pH-induced HT, the reason for the increase in inhibition observed with His-tagged forms of SFV domain III is not known. The tag at the domain III NH2 terminus could act by stabilizing binding to E1, mimicking the important domain I–domain III linker region and/or enhancing its trimeric interactions, concentrating the protein at the membrane at low pH, preventing displacement of the exogenous DIII by the endogenous DIII, and/or preventing cooperative HT–HT interactions. High concentrations of His-tagged DV2 domain III protein did not affect SFV fusion (Fig. 3 B), indicating that there is no nonspecific effect of the His tag.

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