Berkman, A. | bioRxiv Science

bioRxiv2024

Structural and antigenic characterization of novel and diverse Henipavirus glycoproteins

Henipaviruses, a genus within the Paramyxoviridae family, include the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of deadly disease. Recent discoveries of several new Paramyxoviridae species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the Henipavirus and Parahenipavirus genera. Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to here as HNVs, we constructed an expanded, antigenically diverse panel of HNV fusion and attachment glycoproteins from 56 unique HNV strains that better reflects global HNV diversity. We expressed and purified the fusion protein ectodomains and the attachment protein head domains and studied their biochemical, biophysical and structural properties. We performed immunization experiments in mice leading to the elicitation of antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures of diverse fusion proteins elucidated molecular determinants of differential pre-fusion state metastability and higher order contacts. A crystal structure of the Gamak virus attachment head domain revealed an additional domain appended to the conserved 6-bladed, {beta}-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal new cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.

biochemistry↗

bioRxiv2023

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Forster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GPs interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry. Author SummaryEbola virus causes disease in humans with high fatality. A better understanding of how Ebola virus enters cells is critical to inform the development of novel therapeutic and preventative measures. The viral glycoprotein present on the surface of the virus mediates attachment to cells and subsequent entry through a poorly understood mechanism involving fusion of viral and cellular membranes. Here, we employ computational and experimental biophysical techniques to understand how the Ebola glycoprotein senses chemical cues in its environment, such as pH, calcium ions, and specific lipid species to ensure that entry occurs at the right time and place. Our results specify elements of the glycoprotein that control its structure under changing physiological environments.

biophysics↗