A key interaction between the alphavirus envelope proteins responsible for initial dimer dissociation during fusion

Abstract

Alphaviruses such as Semliki Forest virus (SFV) are enveloped viruses whose surface is covered by an organized lattice composed of trimers of E2-E1 heterodimers. The E1 envelope protein, a class II fusion protein, contains the hydrophobic fusion loop and refolds to drive virus fusion with the endosome membrane. The E2 protein is synthesized as a precursor p62, whose processing by furin primes the heterodimer for dissociation during virus entry. Dissociation of the E2-E1 heterodimer is an essential step during low-pH-triggered fusion, while the dissociation of the immature p62-E1 dimer is relatively pH resistant. Previous structural studies described an "acid-sensitive region" in E2 that becomes disordered at low pH. Within this region, the conserved E2 H170 is in position to form a hydrogen bond with the underlying E1 S57. Here we experimentally tested the role of this interaction in regulating dimer dissociation in mature and immature virus. Alanine substitutions of E1 S57 and E2 H170 destabilized the heterodimer and produced a higher pH threshold for exposure of the E1 fusion loop and for fusion of the immature virus. E1 S57K or S57D mutations were lethal and caused transport and assembly defects that were partially abrogated by neutralization of the exocytic pathway. The lethal phenotype of E1 S57K was rescued by second-site mutations at E2 H170/M171. Together, our results define a key role for the E1 S57-E2 H170 interaction in dimer stability and the pH dependence of fusion and provide evidence for stepwise dissociation of the E2-E1 dimer at low pH.

Fig 1

Model for low-pH-triggered conformational changes in the E2/E1 dimer. (A) Schematic model for conformational changes in the E2/E1 heterodimer. (Left panel) During virus biosynthesis, p62, the precursor to E2, is cleaved by furin in the late secretory pathway, allowing E3 release at neutral pH. (Middle panel) During virus entry, exposure to endosomal low pH causes E2 domain B to dissociate (shown as curved arrow), uncovering the E1 fusion loop. (Right panel) The fusion loop inserts into the endosomal membrane, and E1 forms an extended homotrimer. E2 is shown in red with domains A to C indicated and the beta-ribbon connector in pink, E3 is shown in gray, and E1 is shown in blue with domains I to III indicated and the fusion loop in orange. (B) A closeup view of the crystal structure of the CHIK p62/E1 dimer (Protein Data Bank [PDB] entry 3N41) (9) with the proteins colored as described for panel A. E1 S57 and E2 H170 are labeled and represented as sticks in blue and purple, respectively. This figure was prepared using Pymol (47).

Fig 2

Growth kinetics of WT and mutant SFV on CHO and FD11 cells. Control (A) and FD11 furin-deficient (B) CHO cells were infected with WT or mutant viruses at a multiplicity of infection of 0.01 PFU/cell for 90 min at 37°C. After infection, cells were washed twice with serum-free media. At the indicated time postinfection, the virus secreted into the media was collected and the titer was determined by plaque assay on BHK cells. Data represent the results of 2 independent experiments.

Fig 3

pH dependence of fusion of WT and mutant SFV. Mature, E2-containing virus produced by BHK cells (A) and immature, p62-containing virus produced by FD11 cells (B) were adsorbed to BHK cells on ice. Virus-plasma membrane fusion was triggered by treatment at the indicated pH at 37°C for 3 min. Virus-infected cells were then quantitated by immunofluorescence and the results shown as the percentage of maximal fusion for each virus. Entry via endocytosis is low under these conditions, as shown by pH 7.0 controls (see, e.g., panel A). Data represent averages and standard deviations of the results of 3 independent experiments. In Fig. 3B, the difference in the percentage of fusion between WT and E1 S57A-E2 H170A was statistically significant from pH 4.7 to 5.3, and between WT and the mutants E1 S57A and E2 H170A from pH 4.7 to 5.1 (P < 0.05 using a two-tailed t test).

Fig 4

pH dependence of WT and S57A-H170A dimer dissociation. (A) Purified ³⁵S-labeled WT or S57A-H170A virus was treated at the indicated pH values for 10 min on ice. Samples were solubilized in 1% NP-40, adjusted to pH 8.0, and immunoprecipitated using MAb to E1. Samples were analyzed by SDS-PAGE and quantitated by phosphorimaging. The ratio of E2 signal to E1 signal was determined for each pH point and represented as percentage of E2 coprecipitated. Data represent averages and ranges of the results of 2 independent experiments. (B) Purified ³⁵S-labeled WT (black bars) or S57A-H170A (white bars) virus was treated at the indicated pH for 5 min at 37°C. Samples were adjusted to neutral pH and immunoprecipitated with MAb to the E1 fusion loop. Samples were analyzed by SDS-PAGE and quantitated by phosphorimaging and the results shown as the percentage of maximal immunoprecipitation (ranging from 50% to 80% of total E1). Data represent averages and standard deviations of the results of 3 independent experiments. The difference in immunoprecipitation between the WT and E1 S57A-E2 H170A was statistically significant at pH 8, 7, and 6 (P < 0.05 using a two-tailed t test).

Fig 5

Assembly properties of S57K virus. BHK cells were electroporated with WT or mutant viral RNA and incubated at 37°C for 6 h. The cells were then pulse-labeled for 30 min with [³⁵S]methionine-cysteine and chased for the indicated times at 37°C. (A) The cell lysates and media were immunoprecipitated with a polyclonal antibody to the envelope glycoproteins and analyzed by SDS-PAGE. (B) After immunoprecipitation, an aliquot of the lysates was digested with endoglycosidase H as indicated. p62′, E2′, and E1′ indicate the positions of the protein after Endo H digestion. Data represent the results of 2 independent experiments.

Fig 6

Effect of neutralization of the exocytic pH on S57K- and S57D-infected cells. (A) BHK cells were electroporated with WT or mutant viral RNA and incubated at 37°C for 2 h. Medium without (a to c) or with (d to f) 20 mM ammonium chloride was then added to the cells and the incubation continued for an additional 12 h. Cells were then fixed with paraformaldehyde without permeabilization, and indirect immunofluorescence staining was performed with MAb to E1. Fluorescence microscopy images were acquired with the same exposure time. Data represent the results of 3 independent experiments. Bar = 10 μM. (B) Two hours postelectroporation, medium with or without 20 mM ammonium chloride was added to WT or mutant-infected cells. After 4 h at 37°C, the cells were pulse-labeled for 30 min and incubated in chase medium ± 20 mM NH₄Cl for an additional 4 h at 37°C. Chase media were immunoprecipitated with a polyclonal antibody to the envelope glycoproteins in the absence of detergent, and analyzed by SDS-PAGE. Bands labeled with an asterisk represent E1s, a truncated form of E1. Data represent the results of 3 independent experiments.

Fig 7

p62 processing of S57K revertants. BHK cells were infected with WT or mutant virus at a multiplicity of infection of 1 PFU/cell for 90 min at 37°C. After a 5-h incubation, cells were pulse-labeled for 30 min with [³⁵S]methionine-cysteine and chased for 0 (A) or 1 (B) h. Cell lysates were immunoprecipitated with a polyclonal antibody to the envelope glycoproteins and analyzed by SDS-PAGE. Revertants are referred to by the amino acid in the wt SFV sequence, its position, and the revertant amino acid replacement. Data represent the results of 2 independent experiments.

Fig 8

pH dependence of fusion of S57K revertant viruses. Virus stocks were produced in BHK cells. Serial dilutions of virus were adsorbed to BHK cells for 90 min on ice. Virus-plasma membrane fusion was triggered by treatment at the indicated pH at 37°C for 3 min. Virus-infected cells were then quantitated by immunofluorescence and the results shown as the percentage of maximal fusion for each virus. Data represent averages and standard deviations of the results of 3 independent experiments.