Periodic Reporting for period 4 - NanoVirus (Deciphering virus-host interactions using correlated confocal-atomic force microscopy)
Okres sprawozdawczy: 2022-07-01 do 2022-12-31
Viruses are strict intracellular parasites and, because of their simplicity, they depend on a host organism in virtually every stage of their life cycle. Over millions of years of evolution and adaptation to their hosts, they have acquired the molecular "keys" or "entry tickets" necessary to be able to exploit and control cellular functions. As a result, most viruses become species-specific, and related viruses typically infect only a narrow range of plants, animals, bacteria, or fungi. Virus entry pathways are largely defined by preliminary interactions between virus particles and their receptors on the cell surface. Receptor-mediated virus entry is a complex multi-step process in which viruses face fundamental challenges in order to hijack the host machinery. Elucidation of the complex interaction between viruses and their receptors is an important challenge that needs to be addressed if we are to gain a complete understanding of the invasion process.
Considerable efforts have been made to characterize the cellular receptors and pathways that allow virus internalization. Nevertheless, the dynamics of molecular processes in virus-receptor interactions and virus internalization were very poorly understood. The NanoVirus project aimed to develop an innovative microscopic platform to decipher viral infection on living cells at the nanoscale. At the crossroads of nanotechnology, biophysics, cell biology and virology, the main objective of this project was to integrate atomic force microscopy (AFM) with a new generation of high-resolution confocal microscopes to allow us to address complex biological questions. During the project, we studied the molecular mechanisms of viral infection in living mammalian cells. As a prototype of non-enveloped viruses, we first focused on mammalian orthoreoviruses (T3 prototype reovirus), the prototype of the large family Reoviridae, and then extended the study to other viruses such as herpes virus and SARS-CoV-2. We have shown that the early binding of viruses to cell surfaces is a highly regulated process with many different receptors involved in the early binding steps. We were able to uncover cooperative steps between different receptors and also discovered ways to block certain key steps thus opening the way to therapeutic applications.
Considerable efforts have been made to characterize the cellular receptors and pathways that allow virus internalization. Nevertheless, the dynamics of molecular processes in virus-receptor interactions and virus internalization were very poorly understood. The NanoVirus project aimed to develop an innovative microscopic platform to decipher viral infection on living cells at the nanoscale. At the crossroads of nanotechnology, biophysics, cell biology and virology, the main objective of this project was to integrate atomic force microscopy (AFM) with a new generation of high-resolution confocal microscopes to allow us to address complex biological questions. During the project, we studied the molecular mechanisms of viral infection in living mammalian cells. As a prototype of non-enveloped viruses, we first focused on mammalian orthoreoviruses (T3 prototype reovirus), the prototype of the large family Reoviridae, and then extended the study to other viruses such as herpes virus and SARS-CoV-2. We have shown that the early binding of viruses to cell surfaces is a highly regulated process with many different receptors involved in the early binding steps. We were able to uncover cooperative steps between different receptors and also discovered ways to block certain key steps thus opening the way to therapeutic applications.
In the frame of this ERC, we first were able to demonstrate that viral infection is a multiple steps process which involved the cooperativity of multiple cell surface receptors. In particular, we evidenced, for reoviruses, that the first binding step to cell surface-exposed sialic acid (a particular glycan present at the cell surface) serves as a switch to further engage specific entry receptors (Koehler et al, Nat. Commun. (2019)), leading in turn to the specific recruitment of clathrin (Koehler et al, Nat. Commun (2021)), activating the virus entry within the host cell. This observation was possible due to the development of a new biophysical platform in virology which combines atomic force microscopy and high-resolution confocal microscopy (Renard et al., Nat. Commun. (2020)).
The crucial role of glycans during the virus early binding steps was also demonstrated for the herpes virus, an enveloped virus (for a review of the role of glycans in viral infection see Ann. Rev. Virol. (2020)). We introduced a new method to force-probe multivalent attachment directly on living cells, and we showed, for the first time, direct evidence of a new mechanism by which herpesvirus surface glycoprotein acts as a key negative regulator in the first step of binding to cell surface glycans. This work published in Science Advances (2018), shed new light on the infection mechanism and how the virus itself regulates its binding toward controlling the entry and release scenarios and its diffusion at the cell surface.
Our findings, underlying the crucial role of attachment factor to control the infectivity of viruses, provided us with a unique opportunity to manipulate virus binding efficiency and infectivity for vaccine and oncolytic applications. In this context, we filled in a patent (EP19152640.9).
In response to the Covid-19 pandemic, we also investigated how SARS-CoV-2 interacts with host cells. In a first study (Yang et al, Nat. Commun, 2020), a few months after its emergence, we were able to characterize the kinetic and thermodynamic properties of the spike protein interaction with the ACE2 receptor both in vitro and in a cellular context. This study was a milestone in the field and is among the 50 most downloaded papers in the journal Nature Communications in 2020. Based on this study, we then monitored the impact of the new variants on this binding step and at the end of 2021 (Koehler et al., Nat. Commun., 2021), we showed that the mechanisms of the new variants and in particular the Kappa variant (close to Delta) showed a new binding mechanism that could notably reduce the efficacy of the first version of the vaccine. Based on our initial study on SARS-CoV-2 characterizing the binding of SARS-CoV-2 in the cellular context, we observed that, apart from ACE2, other binding events occurred very rapidly. We were able to show that sialic acids, an in particular 9-O-acetylated sialic acids, played an important role in early binding events. We then developed a multivalent molecule with multiple copies of this sialic acid and demonstrated that this molecule was very effective in preventing SARS-CoV-2 binding. We then showed that it significantly reduced the infection of cells by the virus (Petitjean et al, Nat. Commun. 2022). This molecule has been patented (EP21195975.4) and we are currently conducting, in collaboration with the University of Liège (Belgium), tests in mice which suggest promising activity in vivo.
The crucial role of glycans during the virus early binding steps was also demonstrated for the herpes virus, an enveloped virus (for a review of the role of glycans in viral infection see Ann. Rev. Virol. (2020)). We introduced a new method to force-probe multivalent attachment directly on living cells, and we showed, for the first time, direct evidence of a new mechanism by which herpesvirus surface glycoprotein acts as a key negative regulator in the first step of binding to cell surface glycans. This work published in Science Advances (2018), shed new light on the infection mechanism and how the virus itself regulates its binding toward controlling the entry and release scenarios and its diffusion at the cell surface.
Our findings, underlying the crucial role of attachment factor to control the infectivity of viruses, provided us with a unique opportunity to manipulate virus binding efficiency and infectivity for vaccine and oncolytic applications. In this context, we filled in a patent (EP19152640.9).
In response to the Covid-19 pandemic, we also investigated how SARS-CoV-2 interacts with host cells. In a first study (Yang et al, Nat. Commun, 2020), a few months after its emergence, we were able to characterize the kinetic and thermodynamic properties of the spike protein interaction with the ACE2 receptor both in vitro and in a cellular context. This study was a milestone in the field and is among the 50 most downloaded papers in the journal Nature Communications in 2020. Based on this study, we then monitored the impact of the new variants on this binding step and at the end of 2021 (Koehler et al., Nat. Commun., 2021), we showed that the mechanisms of the new variants and in particular the Kappa variant (close to Delta) showed a new binding mechanism that could notably reduce the efficacy of the first version of the vaccine. Based on our initial study on SARS-CoV-2 characterizing the binding of SARS-CoV-2 in the cellular context, we observed that, apart from ACE2, other binding events occurred very rapidly. We were able to show that sialic acids, an in particular 9-O-acetylated sialic acids, played an important role in early binding events. We then developed a multivalent molecule with multiple copies of this sialic acid and demonstrated that this molecule was very effective in preventing SARS-CoV-2 binding. We then showed that it significantly reduced the infection of cells by the virus (Petitjean et al, Nat. Commun. 2022). This molecule has been patented (EP21195975.4) and we are currently conducting, in collaboration with the University of Liège (Belgium), tests in mice which suggest promising activity in vivo.
Results obtained in the frame of NanoVirus contributed to develop a new technique in virology that enables us to address the molecular mechanism by which individual viruses interact with cell surface molecules. This new approach enables to dissect the various steps leading to the virus entry and the role of the individual factors and receptors into these steps. This fine characterization of the early binding steps has shown to be useful to help in developing and testing of new generations of anti-viral molecules.
Given the health context of these last years, the development of this technique before the pandemic allowed us to be directly active in this current issue. We have been able to contribute to the understanding of the molecular mechanism of binding of SARS-CoV-2 to cellular receptors and we have also been able to develop an antiviral molecule that shows promising in vivo activities.
Given the health context of these last years, the development of this technique before the pandemic allowed us to be directly active in this current issue. We have been able to contribute to the understanding of the molecular mechanism of binding of SARS-CoV-2 to cellular receptors and we have also been able to develop an antiviral molecule that shows promising in vivo activities.