Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2

Abstract

The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion.

Keywords: GCS1; HAP2; Tetrahymena; conjugation; evolution of sex; membrane fusion; structure homology modeling; virus fusogen.

Figure 1. Conjugation leads to rapid exchange of labeled cytosolic proteins in mating T. thermophila

Cells of different mating types were labeled with either carboxyfluorescein diacetate succinimidyl ester (CFSE) or Cell Trace™ Far Red (CTFR) and examined microscopically after fixation. Scale bars in all micrographs = 10 μm. (A,B) Overlay of phase and fluorescence images of labeled and unlabeled cells combined and fixed 15 min post-mixing showing either green or red labeling (but no exchange of fluorescent proteins). (C,D) Fluorescence images of labeled and unlabeled partners combined and fixed at 3.5 and 2.5 h post-mixing, respectively. Partial exchange of fluorescent proteins from labeled (bright) to unlabeled (faint) mating partners is seen. (E–H) Fluorescence Image of a wild type pair of cells in which both mating partners were separately labeled, combined and fixed 3.5 h post-mixing. Reciprocal exchange of labeled proteins is visible in the same mating pair viewed with either red (E) or green filter (F) filter sets. (G) Merged image of (E) and (F). (H) Phase image of the mating pair in (E–G). (I–J) Fluorescence images of labeled wild type cells 20 h after mixing. At this time point, pairs have come apart, but exconjugant progeny cells maintain a combination of the parental fluorescent markers. (K) Merged image of (I) and (J). (L) Phase image of the cells in (I–K). (M–P) A cross between ΔHAP2 partners of different mating types with the same sequence of images as in (E–H). Note the absence of fluorescent protein transfer. (Q) Representative data from one experiment showing the kinetics of pairing for WT × WT (◆) and WT × ΔHAP2 (□) crosses. (R) The kinetics of fusion in WT × WT (◆), WT × ΔHAP2 (□) and ΔHAP2 x ΔHAP2 (o) crosses determined as the percentage of pairs showing visible transfer of fluorescent material at the indicated time points. Data are expressed as the mean +/− SEM for 3 and 4 independent experiments (◆ and □, respectively), and for 1 experiment (o).

Figure 2. (see also Figure S1). Quantitation of T. thermophila sexual cell fusion events by flow cytometry

WT and/or ΔHAP2 cells of different mating types were labeled with either CFSE or CTFR, mixed at a 1:1 ratio, and acquired at different time points after mating and fixation. Superscripts (α and β) denote mating types VII and VI, respectively. (A) Representative forward scatter (FSC) / side scatter (SSC) plot showing the distribution of cell size versus granularity of a T. thermophila mating culture and the gate (circled) chosen for further analysis. (B) A flow cytometry plot of labeled WT^α × WT^β (WT^{α × β}) cells fixed 15 min after mixing the different mating types. (C) The same culture as in (B), but instead fixed 16 h after mixing. (D) Representative plot of a WT^α × ΔHAP2^β cross 16 h after mixing. (E) Flow cytometry plot of a ΔHAP2 ^α × ΔHAP2 ^β (ΔHAP2 ^{α × β}) cross 16 h after mixing showing an absence of events in the mid-fluorescence gate. Numbers adjacent to the outlined areas in (A–E) indicate the percent of cells in these gates. (F) Representative histogram comparing the relative size of individual events (based on forward scatter) in the double-labeled CFSE^hi/CTFR^hi gate (grey) versus the single-labeled CTFR^hi gate (red). Median FSC intensity values for these two populations are shown in the inset. (G) The cumulative results of independent mass mating experiments including all biological replicates for different WT, ΔHAP2, and complementation strain crosses (circles represent the percentage of exconjugant cells in the mid-fluorescence gate from individual matings 16 h after mixing; bar represents mean and error bars +/− s.d.). “Genomic” or “cDNA” strains are designated according to which HAP2 gene product was used to complement the ΔHAP2 cell line during their construction. A one-sided Kruskal-Wallis test with a Dunn’s post-test found a significant difference (**** = p<0.0001) between the WT ^{α × β} cross and WT × ΔHAP2 crosses, but no difference (ns= not significant) between the WT ^{α × β} cross and the genomic or cDNA complementation crosses. (H) Flow cytometry plots of cell populations from mated (dark grey) and unmated (red or green) cultures at the 16 h time point shown superimposed. Populations of double-labeled cells from the mated cultures are denoted (F), and single-labeled cells that had undergone “co-stimulation” but had not exchanged fluorescent protein are denoted (CS). Note that the populations with the highest fluorescence intensities (MFI) are the single-labeled starved cells (S) from the cultures that had not been mated. The formulas used to measure the “Percent MFI Gained” (due to transfer of labeled protein from the opposite mating partner) and the “Percent MFI Lost” (due to co-stimulation) are indicated on the right. The calculations are color-coded to show the red or green MFI measurement that was used for each population (S, CS, F). (I) Chart showing the mean +/− s. d. of the percent MFI gained in each mating partner of a given cross, based on the upper formula in (H). The percent MFI gained in F populations was measured with respect to the MFI of the corresponding CS populations, as the co-stimulated partners would theoretically represent the starting fluorescence intensity prior to cellular fusion. Note that no substantial differences were seen in the amount of fluorescent protein exchanged between mating partners in WT × WT and ΔHAP2 × WT crosses. (J) Chart showing the mean +/− s. d. of the percent MFI lost in each mating type of a given cross based on the lower formula in (H). Regardless of the parental cell lines used, a consistent reduction in the MFI was seen in mated cells that had not undergone fusion (CS) when compared to unmated starved cells (S).

Figure 3. (see also Figure S2 and Table S1). Homology modeling predicts a structural similarity between HAP2 and class II viral fusogens

The T. thermophila HAP2 primary sequence was submitted to template-based structural modeling platforms, Phyre2 and RaptorX. Known and predicted structures shown in panels (A–C) are colored by domain according to the convention used for class II fusion proteins: red = domain I; yellow = domain II (with black circles highlighting the known and predicted fusion loops); and, blue = domain III. (A) The known structure of the Dengue Virus envelope glycoprotein ectodomain (DENV, PDB ID: 1UZG)[16]. (B) The Phyre2-predicted partial structure of the T. thermophila HAP2 ectodomain based on the template shown in (A). (C) The RaptorX-predicted T. thermophila HAP2 ectodomain structure based on the Rift Valley Fever Virus Glycoprotein C template (PDB ID: 4HJ1)[17]. (D) Alignment of primary and secondary structural elements in the region of homology between the T. thermophila HAP2 ectodomain and Dengue Virus Envelope generated by Phyre2. Sequence identities are shaded gray. Secondary structural elements are shown on the top line with α-helices indicated by green spirals, and β-strands by blue arrows. The boxed region is the T. thermophila HAP2 sequence aligning to the viral envelope protein’s fusion loop. (E) A table of 17 HAP2 orthologs from other species with the highest confidence hits to class II viral fusogens based on Phyre2 batch processing results (Top class II viral hits are listed as: % confidence, PDB ID of template envelope protein structure, and viral origin: DENV= Dengue Virus, TBEV= Tick Borne Encephalitis Virus, WNV= West Nile Virus).

Figure 4. (see also Figure S3). Sequence elements important for T. thermophila HAP2 function

Mutant cell lines carrying altered versions of the HAP2 gene were mated with a wild type (WT) partner, and the percentage of cells undergoing fusion determined by flow cytometry. Cell lines that showed levels of fusion equivalent to WT ^{α × β} crosses (~80%) were considered to express functional HAP2. (A) Diagrams of truncations / mutations to the ectodomain. The region in and around the predicted fusion loop is expanded, and amino acids targeted for mutations are highlighted in black, while those deleted in the fusion loop truncation are shown in bold black, italicized lettering. (B) Diagrams of truncations / mutations to the cytosolic domain. The region in and around the poly-basic stretch (underlined) is expanded, and the potentially palmitoylated cysteine residues targeted for mutations are highlighted in black. The numbers in (A) and (B) refer to the numerical positions and/or range of truncated amino acids relative to the full-length HAP2 protein sequence. Abbreviations are: SP = signal peptide; DENV = Phyre2-predicted Dengue Virus envelope protein region of homology; FL = fusion loop; H/G = HAP2/GCS1 domain; TM = transmembrane domain; B = poly-basic domain; HA = influenza hemagglutinin epitope tag; FLAG-10xHis = epitope tag. (C, D) Bar charts showing the mean percentage +/− s.d. of exconjugant cells in the mid-fluorescence gate (cells that had undergone fusion) after mating as determined by flow cytometry. Circles represent fusion data from individual matings 16 h after mixing for the various constructs. A one-sided Kruskal-Wallis with Dunn’s post test found no significant differences between the WT ^{α × β} cross (Fig. 2G) and the HAP2 mutant crosses Δ510–513, HAP2 FL Rescue, FQY131-3AAA, R164A, LNL171-3AAA, C5→S, ΔBasic Domain, and ΔC’ term. A modest, yet statistically significant reduction (p=0.0011) in the percentage of fusion was observed for C8→S mutants when compared with the WT ^{α × β} cross. Likewise, no significant differences were found between the WT^α × ΔHAP2^β cross (shown in Fig. 2G) and the WT × HAP2 mutant crosses ΔHAP2 domain, ΔDENV region, ΔFusion Loop, DENV FL Rescue, and CC147-8SS. Sample sizes for each cross are listed in Supplemental Experimental Procedures.

Figure 5. (see also Figure S4). Interaction of the T. thermophila HAP2 fusion peptide with model membranes. (A)

Amino acid sequences of synthetic peptides used in biophysical assays (HAP2 = predicted wild type T. thermophila HAP2 fusion loop; DENV WT = wild type Dengue Virus fusion loop; DENV W101A = mutant version of the DENV peptide with reduced fusogenic activity; Influenza WT = wild type Influenza virus fusion peptide; Influenza G1V = mutant version of the influenza virus fusion peptide with reduced fusogenic activity; R1, R2 = randomized control peptides for the T. thermophila HAP2 fusion loop). Amino acid substitutions that reduce fusogenic activity of the mutant viral peptides are indicated in purple letters. All peptides contained a flexible and polar GGGKKKK tag at their C-termini (not shown)[30]. (B) Circular dichroism spectra of the DENV and predicted Tetrahymena HAP2 fusion loop peptides (2 μg/mL; pH 5) in the presence (thick line) or absence (thin line) of small unilamellar vesicles. (C) The head group (DPPTC) and (D) acyl chain (5PC) spin-labeled lipids (left), are shown next to their corresponding electron spin resonance (ESR) plots (right). ESR plots depict the order parameter (S₀) of spin-labeled lipids within multilamellar liposome vesicles (y-axis) plotted as a function of increasing peptide to lipid ratio (x-axis). Data points and error bars represent the mean +/− s.d. for 2 (DENV W101A); 3 (DENV WT and HAP2); or 3 (R1; R2) independent experiments. (E) Raw data from a representative lipid mixing experiment showing R18 fluorescence dequenching over time. Synthetic fusion peptides were added to a mixed population of R18-quenched and unlabeled liposomes at ~2 min, followed by Triton X-100 at 7–8 min to establish maximum dequenching values for normalization purposes. (F) Bar chart showing the mean and s.d. (error bars) for normalized percent lipid mixing data from 3 independent experiments. All measurements were made at 25°C and membrane compositions consisted of POPC:POPG:Chol=5:2:3.