Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.

Zhu Si,Ming Luo,Ivo Atanasov,Weike Li,Jiayan Zhang,Wong H Hui,Xuekui Yu,Sakar Shivakoti,Chang-Lu Tao,Guo-Qiang Bi,Kang Zhou,Z Hong Zhou

doi:10.1371/journal.ppat.1007452

Abstract

Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the “postfusion” gB ectodomain. We also observed prefusion gB in complex with an “L”-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain.

Highlights

Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae subfamily of the Herpesviridae family, is a leading viral cause of birth defects [1, 2] and a major contributor to lifethreatening complications in immunocompromised individuals
HCMV infection begins with cellular membrane fusion, a dynamic process involving receptor-binding to gH/gL complexes and drastic transformation of fusion protein glycoprotein B (gB) trimer from the metastable prefusion conformation to the stable postfusion conformation
We have used cryo electron tomography incorporating cutting-edge technologies to observe the three-dimensional structures of gB and gH/gL in their native environments of HCMV particles

Summary

Introduction

Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae subfamily of the Herpesviridae family, is a leading viral cause of birth defects [1, 2] and a major contributor to lifethreatening complications in immunocompromised individuals. Receptor-binding to gH/gL-containing complexes— the composition of which differs among clinical and laboratory-adapted HCMV strains and across different herpesviruses [5]—triggers conformational changes of fusion protein gB, leading to fusion of the viral envelope with cell membrane [6]. This use of both a fusion protein and a receptor-binding complex for herpesvirus entry differs from many other enveloped viruses, which use a single protein for both receptor binding and membrane fusion

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