Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion - PubMed (original) (raw)
Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion
Maofu Liao et al. J Cell Biol. 2005.
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.
Figures
Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
Figure 4.
SFV E1 domain III proteins inhibit alphavirus infection in the endocytic pathway. SFV, SIN, VSV, and DV2 were diluted in medium of pH 7.2, containing the indicated concentrations of SFV domain III proteins and incubated with BHK cells for 1 h at 20°C to allow endocytic uptake. Infection was blocked by addition of medium containing NH4Cl, and infected cells were quantitated by immunofluorescence. Data are shown as a percentage of control infection in the absence of domain III proteins. Error bars are the mean ± SD. n = 3.
Figure 5.
SFV E1 domain III proteins inhibit the lipid mixing step of fusion. (A) Fluorescence scan of pyrene-labeled SFV fused with BHK cells. Pyrene-labeled SFV was prebound to BHK cells and incubated at 37°C for 1 min in pH 7.4 medium without domain III protein (curve a), in pH 5.5 medium without domain III protein (curve b), or in pH 5.5 medium with 1 (curve c), 5 (curve d), or 8 μM (curve e) His-DIIIS. Background fluorescence from cells alone was subtracted and the fluorescence emission was normalized for each sample by setting the monomer peak at 397 nm to 5 (arbitrary units). Representative example of three experiments. (B) Comparison of inhibition of lipid mixing by domain III proteins. The fusion between pyrene-labeled SFV and BHK cells was assayed as in A, in the presence of the indicated concentrations of domain III proteins. The difference between the Ex/M at pH 7.4 and after treatment at pH 5.5 without domain III proteins was defined as 100% (control). The difference between the ratios of the pH 7.4 sample and each experimental sample was determined and expressed as a percentage of this control difference. Error bars are the mean ± SD. n = 3.
Figure 6.
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.
Figure 7.
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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