Role of conserved histidine residues in the low-pH dependence of the Semliki Forest virus fusion protein

Abstract

A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.

FIG. 1.

Locations of conserved histidine residues on the SFV E1 protein. The prefusion structure of the SFV E1 ectodomain is shown (PDB accession number 2ALA) (26, 36), with the nomenclature of ß-strands and other structural features taken from reference and . The locations and residue numbers of all E1 histidine residues are indicated, with the conserved histidines shown in green. DI is shown in red, DII is shown in orange (the extension containing the hydrophobic fusion peptide loop) and yellow (the extension containing the ij loop), the DI-DIII linker is shown in gray, and DIII is shown in blue. The positions of the internal fusion peptide loop (FL), the DII ij loop, and the DI-DII hinge region are indicated. The E1 stem and TM regions are missing from this ectodomain structure. This figure was prepared using the PyMOL program (8). N-ter, N terminus.

FIG. 2.

Growth properties of wt and mutant SFV. BHK-21 cells were electroporated with wt or mutant virus RNA and incubated at 37°C. The cell media were collected at the indicated times after electroporation, and virus in the media was quantitated by plaque assay (A and B) or infectious-center assay (C) on BHK-21 cells. Data are the averages of the results from two independent experiments, and ranges are indicated by error bars.

FIG. 3.

Assembly properties of wt and mutant SFV. BHK-21 cells were electroporated with wt or mutant virus RNA, incubated at 37°C for 6 h, pulse labeled with [³⁵S]methionine-cysteine, and chased for the indicated times at 37°C. The cell lysates (A) and media (B) were immunoprecipitated using a polyclonal antibody to the envelope proteins and analyzed by SDS-PAGE. Medium samples were immunoprecipitated in the absence of detergent to allow recovery of intact virus particles containing the viral nucleocapsid. The positions of the envelope (E1 and E2) and capsid (C) proteins are indicated on the right. Shown is an example representative of three experiments.

FIG. 4.

Cell-cell fusion activities of wt and mutant SFV. BHK-21 cells were electroporated with wt or mutant virus RNA, diluted 1:20 with nonelectroporated cells, and cultured overnight at 28°C. The cells were treated with media of the indicated pHs at 37°C for 1 min and incubated at 28°C for 3 h. The number of nuclei per envelope protein-expressing cell was evaluated, and the fusion index was calculated as described in the Materials and Methods. Data are the averages of the results from two independent experiments, and ranges are indicated by error bars.

FIG. 5.

pH dependence of wt and mutant virus-membrane fusion. Virus stocks were incubated with BHK-21 cells on ice for 90 min to permit virus-cell binding and then treated with media of the indicated pHs for 1 min at 37°C to trigger virus fusion with plasma membrane. The cells infected due to low-pH-induced fusion were quantitated by immunofluorescence, and results are shown as percentages of maximal fusion for each virus. Data are the averages of the results from two independent experiments, and ranges are indicated by error bars.

FIG. 6.

pH dependence of wt and H3A mutant dimer dissociation and E1-membrane interaction. (A) E2/E1 dimer dissociation. Purified ³⁵S-labeled wt or H3A virus was mixed with liposomes, treated at the indicated pHs for 5 min at 20°C, and adjusted to pH 7.0. Samples were solubilized in 1% Triton X-100, digested with trypsin for 10 min on ice, and analyzed by SDS-PAGE. The E2 protein in each sample was quantitated by phosphorimaging and expressed as a percent of the total E2 protein in control samples incubated with premixed trypsin and soybean trypsin inhibitor. Data in panel A are the averages of the results from three independent experiments, and standard deviations are shown as error bars. (B) Virus-liposome association. Purified ³⁵S-labeled wt or H3A mutant SFV was mixed with liposomes, treated at the indicated pHs for 30 s at 37°C, and adjusted to pH 8.0. Virus-liposome association was analyzed by cofloatation assay on discontinuous sucrose gradients and expressed as a percentage of the total virus in the gradient. Data in panel B are the averages of the results from two independent experiments, and the ranges are indicated by error bars.

FIG. 7.

pH dependence of E1 homotrimer formation for wt and H3A mutant SFV. Purified ³⁵S-labeled wt or H3A virus was mixed with liposomes, treated at the indicated pHs for 5 min at 20°C, and adjusted to pH 7.0. (A) Aliquots of the samples were solubilized in SDS sample buffer at 30°C, and the SDS-resistant E1 homotrimer bands were quantitated by SDS-PAGE and phosphorimaging. (B) Aliquots of the samples were digested with trypsin for 10 min at 37°C and the trypsin-resistant E1 homotrimers were quantitated by SDS-PAGE and phosphorimaging. Data shown are averages of the results from two independent experiments, with ranges indicated by error bars (A), or three independent experiments, with standard deviations shown as error bars (B).