Cryo-EM structure of the prefusion state of canine distemper virus fusion protein ectodomain

David Kalbermatter,Neeta Shrestha,Flavio M Gall,Marianne Wyss,Rainer Riedl,Philippe Plattet,Dimitrios Fotiadis

doi:10.1016/j.yjsbx.2020.100021

Abstract

Measles virus (MeV) and canine distemper virus (CDV), two members of the Morbillivirus genus, are still causing important global diseases of humans and animals, respectively. To enter target cells, morbilliviruses rely on an envelope-anchored machinery, which is composed of two interacting glycoproteins: a tetrameric receptor binding (H) protein and a trimeric fusion (F) protein. To execute membrane fusion, the F protein initially adopts a metastable, prefusion state that refolds into a highly stable postfusion conformation as the result of a finely coordinated activation process mediated by the H protein. Here, we employed cryo-electron microscopy (cryo-EM) and single particle reconstruction to elucidate the structure of the prefusion state of the CDV F protein ectodomain (solF) at 4.3 Å resolution. Stabilization of the prefusion solF trimer was achieved by fusing the GCNt trimerization sequence at the C-terminal protein region, and expressing and purifying the recombinant protein in the presence of a morbilliviral fusion inhibitor class compound. The three-dimensional cryo-EM map of prefusion CDV solF in complex with the inhibitor clearly shows density for the ligand at the protein binding site suggesting common mechanisms of membrane fusion activation and inhibition employed by different morbillivirus members.

Highlights

Despite the availability of an efficient vaccine, measles virus (MeV) is still causing outbreaks worldwide that are associated with major childhood morbidity and mortality in developing countries (Simons et al, 2012)
The fusion protein dramatically refolds to fuse the envelope of the virus with the host cell plasma membrane, which allows for the injection of the viral nucleocapsid into the host cell cytoplasm
To achieve proper expression yield of Measles virus (MeV) soluble CDV F ectodomain (solF), Hashiguchi and colleagues employed a “locked-stalk” approach that is based on the introduction of supplementary stabilizing disulfide bridges within the stalk region (Hashiguchi et al, 2018); a strategy recently proven to be effective in case of stabilizing the prefusion state of the respiratory syncytial virus F protein (Stewart-Jones et al, 2015)

Summary

Introduction

Despite the availability of an efficient vaccine, measles virus (MeV) is still causing outbreaks worldwide that are associated with major childhood morbidity and mortality in developing countries (Simons et al, 2012). MeV and CDV F protomers are first synthesized as long inactive precursors, which must be cleaved by furin proteases residing within the Golgi apparatus to achieve trimeric fusion-competent, disulfide-linked, F1 and F2 subunits Such primed trimeric F complexes are characterize by a high energy-containing state (i.e., metastable), which is commonly referred to as the prefusion conformation. To achieve proper expression yield of MeV solF, Hashiguchi and colleagues employed a “locked-stalk” approach that is based on the introduction of supplementary stabilizing disulfide bridges within the stalk region (Hashiguchi et al, 2018); a strategy recently proven to be effective in case of stabilizing the prefusion state of the respiratory syncytial virus F protein (Stewart-Jones et al, 2015). By employing cryo-electron microscopy (cryo-EM) and single particle reconstruction, we successfully determine the three-dimensional (3D) structure of prefusion CDV solF at 4.3 Å resolution

Expression and purification of CDV solF in the presence of 3G

CDV F ectodomain expression and purification

Structure determination and overall structure of CDV solF

Inhibitor binding pocket location and description