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National Biomedical Center
for Advanced Electron Spin Resonance Technology

Our research is supported by a grant from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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Class II Viral Fusogens and the Ancestral Gamete Fusion Protein HAP2 are Linked using ESR Methodology

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 found that a segment of HAP2 ectodomain from Tetrahymena thermophila and other species adopts a protein fold remarkably similar to the fusion peptide (FP) of the Dengue virus E glycoprotein, which is classified as a class II viral fusogen. Previously we used electron spin resonance (ESR) to study the effect of viral FPs on membranes containing spin-labeled lipids. Based upon detailed ESR lineshape analyses using the MOMD model, the incipient membrane-ordering effect of influenza virus and HIV FPs was consistently observed, indicating that the membrane ordering effect, interpreted as a phenomenon associated with membrane dehydration, is a required step for viral membrane fusion. Using this powerful method, we now showed that this putative HAP2 FP also induces membrane ordering in its polar region, and its shallow hydrophobic region as the Dengue Virus FP and influenza FP do, while two peptides with randomly shuffled sequence as the HAP2 FP have no such effect. Vesicle-vesicle lipid mixing experiments also confirmed that the HAP2 FP induces lipid mixing. Together with the biological study using 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, we confirmed the importance of this HAP2 FP in gametic membrane fusion. Thus, the HAP2 FP that is structurally similar to the Dengue virus FP was identified. The results are important to the understanding of the mechanism of gametic membrane fusion, which is less known compared to the viral counterpart. These results also 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.

Funding: P41GM103521, R01EB003150 (to JHF), 2P400D010964-12 (to TGC), NSF Grad. Res. Fellowship DGE-1144153 (to JFP).

Publication: Pinello JF, Lai AL, Millet JK, Cassidy-Hanley D, Freed JH, and Clark TG. Curr. Biol. 27(5), 651-660 (2017); PMCID: PMC5393271.

The hypothesis of the evolutionary relationship between the gametic fusogen & the Class II viral fusogen.
Jennifer Fricke Pinello (Department of Microbiology & Immunology, Cornell University, Ithaca, NY)
Alex Liqi Lai (ACERT)
Jean K. Millet, Donna Cassidy-Hanley (Department of Microbiology & Immunology, Cornell University, Ithaca, NY)
Jack H. Freed (ACERT)
and Theodore G. Clark (Department of Microbiology & Immunology, Cornell University, Ithaca, NY)
June, 2017