Stabilisation of Viral Membrane Fusion Proteins in Prefusion Conformation by Structure-Based Design for Structure Determination and Vaccine Development

Abstract

The membrane surface of enveloped viruses contains dedicated proteins enabling the fusion of the viral with the host cell membrane. Working with these proteins is almost always challenging because they are membrane-embedded and naturally metastable. Fortunately, based on a range of different examples, researchers now have several possibilities to tame membrane fusion proteins, making them amenable for structure determination and immunogen generation. This review describes the structural and functional similarities of the different membrane fusion proteins and ways to exploit these features to stabilise them by targeted mutational approaches. The recent determination of two herpesvirus membrane fusion proteins in prefusion conformation holds the potential to apply similar methods to this group of viral fusogens. In addition to a better understanding of the herpesviral fusion mechanism, the structural insights gained will help to find ways to further stabilise these proteins using the methods described to obtain stable immunogens that will form the basis for the development of the next generation of vaccines and antiviral drugs.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation

“…Even more remarkable is the number of compensatory mutations mapping to gB DV (Figure 4B-C) attributing an important functional relationship between DIII and DV. Recent determination of the pre-fusion structure of HCMV gB shows that DV, by contrast to DIII, undergoes substantial rearrangement during the conformational change [1, 6]. Refolding and extension of DV is needed for the transition of the extended intermediate to the post-fusion conformation.…”

“…Processing by proteolytic cleavage at the furin site [3,4] results in an amino-(~116 kDa) and carboxy-terminal (~58 kDa) fragment, held together by two intramolecular disulfide bonds between cysteine residues C94 and C551 in domain (D)IV, as well as C111 and C507 in DIII [3]. gB is a membrane-bound homotrimer whose trimeric coiled-coils are formed by the α3 helices of DIII, centered on the three-fold axis of the ectodomain generating the trimerization contacts of the protomers [1,[5][6][7]. While comparison of gB pre-and post-fusion structures reveal major structural rearrangements during the transition process, the central core of the protein seems to remain nearly unchanged [6] indicating the importance of this stabilisation for fusion itself.…”

“…Glycoprotein B (gB) is the highly conserved class III fusogen of herpesviruses [1]. The three dimensional conformations of pre- and post-fusion gB structures of different herpesviruses are well conserved [2] despite limited sequence similarity.…”

“…Binding of a gH/gL complex to the host cell receptor brings the viral and cellular membrane to close proximity for the gB fusion loop to insert into the membrane. Here, gB undergoes a major conformational change from a metastable pre-fusion to a stable post-fusion conformation resulting in the merging of the two membranes and the release of the viral content into the cell [1, 6]. Fusion at the plasma membrane of fibroblasts is pH-independent [13] and in the endosome of epithelial, endothelial, and myeloid cells pH-dependent [14].…”

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses

“…The WT Env ectodomain served as the template for introduction of mutations commonly used to stabilize viral fusion proteins in their prefusion conformation [51]: 1) R126A mutation in the furin-like site on the leader peptide prevented the SP/N-SU cleavage [26], 2) the canonical furin site (567-570) was replaced with a flexible and non-cleavable linker (GGGGSGGGGS) abrogating the cleavage, 3) foldon trimerization motif was added at the C-terminus (SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLG). The resulting expression construct was termed variant 1 (V.1 on Fig.…”