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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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Domain III proteins specifically inhibit alphavirus and flavivirus fusion. (A) Inhibition by alphavirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. The cells were washed and cultured in medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection (pH 5.5, no protein). (B) Inhibition by flavivirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. Infected cells were quantitated as in A. (C) Domain III protein does not release DV2 from cells. DV2 was bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min at the indicated pH in the presence or absence of 50 μM DV2DIIIH1. For samples treated at pH 7.4, cells were incubated for 2 h at 37°C and infected cells were quantitated by immunofluorescence as in A. For samples treated at pH 5.7, cell-associated radiolabeled virus capsid protein was quantitated by SDS-PAGE of cell lysates. Average of three experiments. Error bars are the mean ± SD. n = 3.
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fig3: Domain III proteins specifically inhibit alphavirus and flavivirus fusion. (A) Inhibition by alphavirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. The cells were washed and cultured in medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection (pH 5.5, no protein). (B) Inhibition by flavivirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. Infected cells were quantitated as in A. (C) Domain III protein does not release DV2 from cells. DV2 was bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min at the indicated pH in the presence or absence of 50 μM DV2DIIIH1. For samples treated at pH 7.4, cells were incubated for 2 h at 37°C and infected cells were quantitated by immunofluorescence as in A. For samples treated at pH 5.7, cell-associated radiolabeled virus capsid protein was quantitated by SDS-PAGE of cell lysates. Average of three experiments. Error bars are the mean ± SD. n = 3.

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)

Domain III proteins specifically inhibit alphavirus and flavivirus fusion. (A) Inhibition by alphavirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. The cells were washed and cultured in medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection (pH 5.5, no protein). (B) Inhibition by flavivirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. Infected cells were quantitated as in A. (C) Domain III protein does not release DV2 from cells. DV2 was bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min at the indicated pH in the presence or absence of 50 μM DV2DIIIH1. For samples treated at pH 7.4, cells were incubated for 2 h at 37°C and infected cells were quantitated by immunofluorescence as in A. For samples treated at pH 5.7, cell-associated radiolabeled virus capsid protein was quantitated by SDS-PAGE of cell lysates. Average of three experiments. Error bars are the mean ± SD. n = 3.
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Related In: Results  -  Collection

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fig3: Domain III proteins specifically inhibit alphavirus and flavivirus fusion. (A) Inhibition by alphavirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. The cells were washed and cultured in medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection (pH 5.5, no protein). (B) Inhibition by flavivirus domain III proteins. Viruses were bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min under the indicated conditions. Infected cells were quantitated as in A. (C) Domain III protein does not release DV2 from cells. DV2 was bound to BHK cells for 90 min on ice and incubated at 37°C for 1 min at the indicated pH in the presence or absence of 50 μM DV2DIIIH1. For samples treated at pH 7.4, cells were incubated for 2 h at 37°C and infected cells were quantitated by immunofluorescence as in A. For samples treated at pH 5.7, cell-associated radiolabeled virus capsid protein was quantitated by SDS-PAGE of cell lysates. Average of three experiments. Error bars are the mean ± SD. n = 3.
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