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

Date Published: December 3, 2018

Publisher: Public Library of Science

Author(s): Zhu Si, Jiayan Zhang, Sakar Shivakoti, Ivo Atanasov, Chang-Lu Tao, Wong H. Hui, Kang Zhou, Xuekui Yu, Weike Li, Ming Luo, Guo-Qiang Bi, Z. Hong Zhou, Félix A. Rey.

http://doi.org/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.

Partial Text

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 life-threatening complications in immunocompromised individuals. As one of the largest membrane-containing viruses, HCMV shares a common multilayered organization with all other herpesviruses, composed of an icosahedrally ordered nucleocapsid enclosing a double-stranded DNA genome, a poorly defined tegument protein layer, and a pleomorphic, glycoprotein-embedded envelope [3]. During infection, herpesviruses fuse their envelopes with cell membranes, resulting in the delivery of nucleocapsid into the cytoplasm of the host cells. This complex process requires a number of viral glycoproteins and host receptors functioning in a coordinated manner. Glycoproteins gB and gH/gL are conserved across all herpesviruses and are essential for virus entry into cells [4]. 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.

Since the postfusion conformation of gB is energetically favorable and structurally more stable, it is not surprising that purified recombinant gB so far have all adopted the “postfusion” conformation [12, 16,45]. Therefore, imaging gB in its native, virion environment by cryoET seems to be the necessary approach to obtain the in situ structure in its metastable, prefusion conformation. However, a major challenge in interpreting in situ cryoET structures is the intrinsic poor contrast of tomographic reconstructions due to the use of low electron dose in order to avoid radiation damage to specimen. Poor contrast makes it difficult to identify different molecules or structures for subtomographic averaging. Normally for cellular tomography without phase plate, one could image with a large defocus value to achieve better contrast, aiding in distinguishing densities with different characteristics for subtomographic averaging. However, such approach only offers limited improvements in contrast (S1 Fig), and difficulties still exist in identifying the slender gB in postfusion conformation in our tomograms. This experience is consistent with two previous cryoET studies on HSV-1 gB structures, in which large defocus values were used to increase contrast to facilitate subsequent subtomographic averaging, yet the resulting structure either is at much lower resolution [26] than reported here or has led to controversial interpretations [27]. The greatly improved contrast afforded by VPP technology allowed the differentiation of various glycoprotein structures based on their characteristic appearances on the virion membrane (Fig 7A and 7B; S1 Fig). Therefore, cryoET with VPP offers a clear advantage in resolving structures of proteins in the native environments, enabling their identifications and subtomographic averaging to obtain structures of multi-functional states, as also demonstrated by the existence of two states of 26S proteasome inside neurons [31].

 

Source:

http://doi.org/10.1371/journal.ppat.1007452

 

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