Two domains that control prefusion stability and transport competence of the measles virus fusion protein

Abstract

Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.

FIG. 1.

Escape mutants resistant to inhibition cluster predominantly at F residue 462. (A) Mutations found in the F proteins of adapted MV-Edm (I) or MV-KS (II to V) variants compared to the parental strains. Sequence changes in the F protein of a resistant primary isolate (MV-B3-2) are shown in comparison to the related sensitive isolate MV-B1. TM, transmembrane. (B) Transient dose-response assay to determine the contributions of selected mutations to resistance. Individual mutations were rebuilt by site-directed mutagenesis in the F-Edm or F-B3-2 background, respectively, and all F expression plasmids were cotransfected with equal amounts of MV H encoding plasmid of the corresponding genotype (MV H-Edm [left] or MV H-B3-2 [right]). Syncytium formation in the presence of different AS-48 concentrations was quantified and values were normalized for cells treated with solvent (DMSO) only. The means of four replicates are shown. The error bars indicate standard deviations. (C) Quantitative cytopathicity assay to assess resistance in the context of viral infection. Mutants confirmed to confer resistance in transient assays were rebuilt in the MV-Edm genome, recombinant virions were recovered, and virus-induced cytopathicities in the presence of different AS-48 concentrations were determined. For comparison, unmodified rMV-Edm (left) and rMV-Edm harboring F B3-2 (462N) (right) are shown. The values were normalized as described above and represent the means of four replicates.

FIG. 2.

Localization of residue 462 in the final MV F core structure. (A) Ribbon model of the MV 6-HB; N termini of HR-B domains are facing up. For clarity, residue 462 is highlighted in only one of the three HR-B domains. TM, transmembrane. (B) Surface model of the MV 6-HB, shown with only one HR-B ribbon for clarity. Residues V459, L457, and L454 are predicted to interact with a hydrophobic groove in the HR-A trimer. HR-A residue E170 is predicted to engage in hydrogen bonding with residues N462 and N465. (C to F) Enlarged ribbon models of HR-A and HR-B highlighting the interaction described above (C). Dynamic structural modeling predicts disruption of the hydrogen bonding, destabilizing the interaction for either of the resistant mutants based on distance (D), charge (E), or steric hindrance (F). (G and H) MD simulations predict greater destabilization of the mutant 6-HBs compared to the wild type. The mutants increase the peptides' hydrophobic exposure to water through the course of the simulation, indicating greater dissociation (G). Hydrophobic solvent-accessible surface (SAS) areas are shown for 50-ps MD simulation. Hydrophilic exposures to water were similar for the four structures, with average hydrophilic SAS areas over the last 10 picoseconds of simulation between 91.1 nm² and 92.0 nm² (H).

FIG. 3.

Resistance mutations at position 462 reduce the efficiency of 6-HB formation. (A) Peptide competition assay determining the efficiencies of virus inhibition by synthetic HR-B-derived peptides. (Left) Cells were infected with unmodified rMV-Edm or mutant rMV-Edm F (462S) in the presence of increasing concentrations of a modified peptide resembling one of the variants identified (462S; sequence shown above graph), and cell-associated viral titers were determined by TCID₅₀ titration. (Right) Peptide competition assay of all rMV F variants described, using an unmodified synthetic peptide (462N; sequence shown above the graph). Shaded letters in the peptide sequences indicate the position of residue 462. To facilitate comparison of different strains, the values were normalized (norm.) for growth in the absence of compound and represent the means of two experiments. The error bars indicate standard deviations. (B) Quantitative cytopathicity assay to assess resistance of rMV-Edm F (E170A) virions to AS-48. Virus-induced cytopathicities were determined in the presence of different AS-48 concentrations. For comparison, unmodified rMV-Edm is shown. The values were normalized for cells infected in the presence of equal amounts of solvent (DMSO) only and represent the means of four replicates. (C) Coimmunoprecipitation of MV F with HR-B peptide in the presence (+) or absence (−) of AS-48. Cells expressing MV H and F (left) were incubated with 41 μM Flag-tagged peptide (pep) and 100 μM AS-48 or an equal amount of solvent (DMSO) and subjected to immunoprecipitation (IP) using anti-Flag antibodies, and precipitates were analyzed by Western blotting using anti-F tail antibodies. For control, immunoprecipitation was carried out in the absence of peptide. Cells expressing MV F only (right) were otherwise treated and analyzed in the same way.

FIG. 4.

The nature of residue 94 in the F cavity determines the effects of changes of residue 462. (A) Syncytium formation after cotransfection of cells with plasmid DNA encoding MV H-Edm and MV F-Edm or F-Edm variants as indicated. Mock-transfected cells (mock) received MV H encoding plasmid only; the plates were photographed 20 h posttransfection at a magnification of ×200 (shown at ×186). (B) Fusogenicity of F-Edm variants cotransfected with H-Edm. Cytotoxicity as an indicator for the ability to induce syncytium formation was quantified as described for Fig. 1B. The values were normalized for unmodified F-Edm and represent the means of three independent experiments. The error bars indicate standard deviations. (C) Surface biotinylation of cells expressing different F-Edm variants to determine F plasma membrane steady-state levels. Biotinylated proteins were precipitated and separated by SDS-PAGE, and F-antigenic material was detected with specific antisera directed against the cytosolic domain of F. The values are based on densitometric quantification using a VersaDoc system and indicate the average percentage of surface material relative to unmodified F-Edm calculated from three to six independent experiments. Standard deviations are shown in parentheses. Mock-transfected cells received vector DNA only. (D) EndoH of F-antigenic material. The F₀ fractions of both mutants analyzed were sensitive to EndoH treatment (F_deglyc material), indicating an ER-type carbohydrate chain conformation. The asterisk marks the small Golgi fraction of EndoH-resistant F-Edm prior to proteolytic maturation. For control, an F variant (F-ER) carrying a KKXX ER retention signal was included. All samples were analyzed by SDS-PAGE and immunoblotting subsequent to EndoH treatment.

FIG. 5.

F mutants 94G 462S and 94G 462N are temperature sensitive. (A) Surface biotinylation and detection of F-antigenic material, as previously described, after incubation of cells at 30°C or 37°C. The values represent densitometric quantification of surface-expressed material and are expressed as the percentage of unmodified F-Edm found at 30°C or 37°C, respectively. (B) Quantification of syncytium formation of selected F-Edm variants after cotransfection of cells with equal amounts of plasmid DNA encoding MV H and F and incubation at 30°C or 37°C as indicated. The values represent the means of four experiments and are expressed as the percentage of syncytium formation activity observed for unmodified F-Edm after incubation at 30°C or 37°C, respectively. For each F variant, the percent syncytium formation at 30°C compared to the same variant at 37°C is given above the graph. The error bars indicate standard deviations.

FIG. 6.

Compounds OX-1 and AS-48 stabilize a transport-competent conformation of mutant F trimers. (A) Surface expression of F mutants 94G 462S and 94G 462N subsequent to incubation of cells in the presence of 75 μM AS-48 or equal amounts of solvent (DMSO) only. The values represent densitometric quantification of surface material as described previously. (B) Quantification of surface expression of all F double mutants generated subsequent to incubation in the presence or absence of compound AS-48 as detailed in the legend to panel A. The values represent the means of three to six independent experiments, and standard deviations are shown. (C) Syncytium formation after cotransfection of cells with plasmid DNA encoding unmodified F-Edm or the 94G 462S F-Edm variant and MV H-Edm and incubation in the presence of 75 μM AS-48 or equal amounts of solvent (DMSO) only. Mock-transfected cells received H-Edm encoding plasmid only, and the plates were photographed 24 h posttransfection at a magnification of ×200 (shown at ×168). (D) Dose-dependent increase of fusion activity after cotransfection of cells with F variant 94G 462S and H-Edm and incubation in the presence of different concentrations of compound OX-1 or AS-48 as indicated. The values represent three independent experiments and are expressed as percentages of fusion activity induced by unmodified F-Edm in the presence of solvent (DMSO) only. The error bars indicate standard deviations.