Gaudin, Y. | Search bioRxiv

bioRxiv2025

Structures of vesicular stomatitis virus glycoprotein G alone and in complex with a neutralizing antibody

VSV G mediates viral entry via endocytosis. In the endosome, G undergoes a pH-dependent conformational change from pre- to post-fusion state, catalyzing membrane fusion. So far, no complete structure of G has been reported. We report cryo-EM structures of G, isolated from virions using detergent, alone and in complex with neutralizing antibody FAb that binds G in all conformations. The post-fusion structure reveals novel details about the organization of the C-terminal part of the ectodomain, showing that it undergoes conformational rearrangement and stabilizes the post-fusion trimer by nesting into a groove between adjacent fusion domains. The fusion loops are visible inside the micelle, which is not the case of the transmembrane domains, suggesting that they are rather mobile. Structures of G-FAb complex show that the epitope belongs to a conserved antigenic site. This work has potential implications for vaccine development and oncolytic virotherapy.

biochemistry↗

bioRxiv2024

Optimization of the VSV G backbone for amino terminal fusion with nanobodies allowing its retargeting to receptors of therapeutic interest.

Vesicular stomatitis virus (VSV) is a promising oncolytic virus. Additionally, its glycoprotein G is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors for gene therapy. However, G receptors (LDLR family members) are ubiquitous and expressed at the surface of non-target cells, precluding in vivo gene therapy. Recently, we identified G mutants that no longer bind to LDLR but retain their fusion activity. This opened the possibility of specifically retargeting the glycoprotein to receptors of interest. Here, we constructed chimeric glycoproteins fused with a nanobody at the amino-terminus of G. By experimental evolution, we identified two mutations in G improving the folding and functionality of chimeric Gs, regardless of the nanobody inserted at the amino-terminus. We then constructed chimeric glycoproteins using several nanobodies targeting HER2 receptor and, into these chimeras, we introduced mutations that abolish the recognition of LDL receptors. VSV and lentiviruses pseudotyped with these glycoproteins specifically infect cells expressing HER2. We have therefore identified G mutations that optimize the G scaffold to tolerate amino-terminal insertion of a nanobody and establish proof of concept that this approach can be used to confer a new tropism on G. This paves the way for targeted in vivo therapies.

bioengineering↗