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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 SFV fusion with target cell membranes. (A) Exogenous domain III specifically inhibits SFV fusion. SFV was added to BHK cells (multiplicity of infection ∼0.002) for 90 min on ice (Binding). The cells were incubated at pH 7.4 (N) or pH 5.5 for 1 min at 37°C to induce fusion (Fusion) and cultured at 28°C overnight in medium containing 20 mM NH4Cl (Culture). The presence or absence of 4 μM His-DIII in each step is indicated by + or −. Infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection in the absence of His-DIII at any step. Representative example of two experiments. (B) The concentration dependence of inhibition by domain III proteins was determined using the assay in A and adding the indicated concentrations of domain III proteins only during the 1-min low pH treatment. Representative example of two experiments.
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fig2: SFV E1 domain III proteins inhibit SFV fusion with target cell membranes. (A) Exogenous domain III specifically inhibits SFV fusion. SFV was added to BHK cells (multiplicity of infection ∼0.002) for 90 min on ice (Binding). The cells were incubated at pH 7.4 (N) or pH 5.5 for 1 min at 37°C to induce fusion (Fusion) and cultured at 28°C overnight in medium containing 20 mM NH4Cl (Culture). The presence or absence of 4 μM His-DIII in each step is indicated by + or −. Infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection in the absence of His-DIII at any step. Representative example of two experiments. (B) The concentration dependence of inhibition by domain III proteins was determined using the assay in A and adding the indicated concentrations of domain III proteins only during the 1-min low pH treatment. 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 E1 domain III proteins inhibit SFV fusion with target cell membranes. (A) Exogenous domain III specifically inhibits SFV fusion. SFV was added to BHK cells (multiplicity of infection ∼0.002) for 90 min on ice (Binding). The cells were incubated at pH 7.4 (N) or pH 5.5 for 1 min at 37°C to induce fusion (Fusion) and cultured at 28°C overnight in medium containing 20 mM NH4Cl (Culture). The presence or absence of 4 μM His-DIII in each step is indicated by + or −. Infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection in the absence of His-DIII at any step. Representative example of two experiments. (B) The concentration dependence of inhibition by domain III proteins was determined using the assay in A and adding the indicated concentrations of domain III proteins only during the 1-min low pH treatment. Representative example of two experiments.
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fig2: SFV E1 domain III proteins inhibit SFV fusion with target cell membranes. (A) Exogenous domain III specifically inhibits SFV fusion. SFV was added to BHK cells (multiplicity of infection ∼0.002) for 90 min on ice (Binding). The cells were incubated at pH 7.4 (N) or pH 5.5 for 1 min at 37°C to induce fusion (Fusion) and cultured at 28°C overnight in medium containing 20 mM NH4Cl (Culture). The presence or absence of 4 μM His-DIII in each step is indicated by + or −. Infected cells were quantitated by immunofluorescence. Results are shown as a percentage of control infection in the absence of His-DIII at any step. Representative example of two experiments. (B) The concentration dependence of inhibition by domain III proteins was determined using the assay in A and adding the indicated concentrations of domain III proteins only during the 1-min low pH treatment. 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