Periodic Reporting for period 1 - HSV1ENTRYPROTEINMAP (Spatial arrangement and interactions of HSV-1 cell entry associated proteins on the native viral envelope)
Reporting period: 2023-03-01 to 2025-02-28
Herpesviruses are a family of large, complex, DNA viruses that are highly prevalent across the global human population, causing life-long infections and cycle between latent and active disease. In contrast to other viruses that rely on a single fusion protein to facilitate viral entry, HSV-1 requires a minimum of four essential virus-encoded glycoproteins to facilitate cell entry . Out of at least twelve different glycoprotein species present on virions, gB, gD, and gH/gL together mediate the process of membrane fusion between the viral and host cell membrane. Although all four glycoproteins are known to be necessary for membrane fusion, the molecular details and stoichiometry of the interactions as well as how the interactions regulate or influence the fusion process remains unknown. Furthermore, despite extensive research in artificial systems no direct evident exists of these interactions on native viruses. From a structural perspective, the ectodomains of these membrane proteins have been determined by X-ray crystallography, and different conformations of the main fusion protein (gB) have been visualised by electron cryotomography (cryoET), however neither structural organisation of individual proteins nor the interactions between them have been investigated on intact HSV-1 envelopes. We presently lack high-resolution understanding (beyond light microscopy) of fundamental questions such as how HSV-1 envelope proteins are arranged in situ, how this effects function and how these changes over time – or specifically during the infection processes. To address this line of questioning our lab has achieved an exciting leap forward in specifically identifying proteins of interest during cryoET imaging by developing signpost origami tags (SPOTs) . The technique of DNA origami was employed to build a “signpost” structure, which consists of a high contrast “sign” and functionalized “post” base linked to a targeting moiety. These SPOTs extend beyond state-of-the-art cryoET to enable the identification of proteins of interest on crowded, pleomorphic biological surfaces. As such they offer unique tools to decipher the functional nanotopology on viral surfaces.
I was able to determine at nanometre resolution the spatial arrangement of essential entry proteins required at the onset of the HSV-1 infection. This already gives us insight into the mechanism of action of viral glycoproteins in their native environment that ultimately leads to HSV-1 fusion. Mapping of the distribution of the entry associated glycoproteins relative to each other and relative to viral structures including capsid was achieved for a test subset of tomograms for gD and gC. The data has been acquired for gB and gH, and expanded for gD and gC for more data points but the processing is ongoing. Future mapping aims to also take into account the glycoprotein distribution relative to the tegument densities.
This research is highly innovative because it was designed to push current technological barriers, by taking advantage of recent methodological advances, including SPOTs, to answer specific key biological questions in herpesvirus entry. This next frontier approach provides a platform to unveil the mechanistic underpinning of the fusion machinery control in an important human pathogen. It allowed determination at nanometer resolution scale the spatial arrangement of essential entry proteins required at the onset of the HSV-1 infection. Methodologically this project has expanded the potential use of SPOTs for identification of proteins in tomography, both within and beyond the current project. We have, for the first time covalently conjugated proteins, including nanobodies, to the DNA origami structures and can show specific targeting of a protein of interest. For the first time we are able to identify a single protein of interest in a complex, multiprotein, native, membrane environment. To maximise the impact of this work we have engaged with others researchers exploring how this system can be implemented for studying a range of membrane associated proteins by cryoET.