Molecular determinants of the SARS-CoV-2 fusion peptide activity

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2), emerged in late 2019 and quickly spread worldwide. SARS-CoV-2 is an enveloped virus and its entry into host cells is mediated by the spike glycoprotein (S-protein) [1]. The S-protein is composed of two subunits (S1 and S2) that contain essential domains for the viral entry mechanism, such as the fusion peptide (FP) which inserts into and disturbs the host cell membrane promoting the fusion between viral and host membranes. Despite its relevance for viral entry, there is still no consensus among scientists for its location on the S-protein and amino acid sequence, although two major candidate regions have been proposed [2, 3]. To shed light on this matter, we combined computational and experimental methods to characterize and compare the effect of the two putative SARS-CoV-2 FPs. We performed a systematic analysis of the SARS-CoV-2 putative FPs, using Molecular Dynamics simulations, to dissect how these peptides interact with the membrane. In parallel, we evaluated the putative FPs behavior in membrane model systems applying biophysical techniques. Since both FPs revealed modest fusogenic activity, we hypothesized that a longer FP or a cooperation among the individual FPs might be required to achieve fusion between viral and host membranes. Given the pivotal role of the FP to viral entry, our work provides relevant insights on the SARS-CoV-2 entry mechanism.

The impact of SARS-CoV-2 Omicron variant on the human ACE2 binding: a computational study

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic, which escalated into a global pandemic in early 2020, accounting for more than 400 million infections and more than 6 million confirmed deaths worldwide (as of 2022/03/10). The SARS-CoV-2 mechanism of transmission and infection involves the binding of the virus to the angiotensin-converting enzyme 2 (ACE2) host receptor through the receptor-binding domain (RBD) of the spike (S) protein. The RBD is a privileged target of our immune system and antiviral therapies. Throughout last year multiple vaccines and new therapeutics against SARS-CoV-2 have been developed. However, their effectiveness is challenged by the continuous evolution of SARS-CoV-2, accompanying the origin and spread of new variants of concern (VOC): Alpha, Beta, Gamma, Delta, and recently, Omicron. Among the reported mutations in the VOC S proteins, several are specific to the RBD, which are associated with higher transmissibility or the ability to escape the immune response of previously infected patients. (Previously published in: Greaney, A.J. et al. (2021) Cell Host Microbe 29,44- 57). In late 2021, the newly SARS-CoV-2 Omicron VOC raised considerable global concern due to the presence of more than 30 mutations in the S protein, 15 of which occur in the RBD (Previously published in: Mannar D et al. (2022) Science 375,760-764). Here we investigated the impact of the VOC RBD mutations on its interaction with ACE2, with a major focus on the Omicron RBD, by performing microsecond molecular dynamics (MD) simulations of this complex. Our analysis of the binding and structural dynamics of these mutations provided a detailed characterization of the binding mode between the VOC RBDs and the receptor. This allowed us to understand the role of key residues in the VOC RBD-ACE2 interface and the effect of specific substitutions on the binding affinity via the establishment of new inter-protein contacts.

Design, production and characterization of antiviral proteins targeting SARS-CoV-2

The virus responsible for the current COVID -19 pandemic is SARS-CoV-2, which has caused >400 million infections and >5 million deaths (as of February 2022). Despite vaccination efforts, there is still an urgent need to develop strategies to control infection and treat patients. One of the proteins bound to the viral membrane is the spike (S) protein, which consists of two subunits: S1, which contains a receptor-binding domain (RBD) responsible for binding to the host cell receptor, and S2, which facilitates membrane fusion between the viral and host cell membranes, previously published in: Jackson CB et al. (2018) Nat Rev Mol Cell Biol 23, 3-20. Thus, this protein is primarily responsible for the ability of the virus to enter host cells, making it one of the most promising therapeutic targets of coronavirus, previously published in: Cao L et al. (2020) Science 6515, 426- 431. The aim of this work was to design and produce antiviral proteins that could prevent the interaction between the two proteins and thus block infection by binding to the RBD region and blocking its interaction with the host receptor, angiotensin converting enzyme-2 (ACE2) protein. First, several antiviral proteins were computationally designed using the Rosetta program based on the interactions between ACE2 and the RBD. Next, six molecular dynamics simulations (MD) of 1 ls of three candidates were performed to test their interaction with the RBD. This was followed by experimental validation after expression and purification of the three candidates. The secondary structure and thermostability of these proteins were tested by far-UV circular dichroism spectropolarimetry. Surface plasmon resonance was used to evaluate the affinity of each candidate for RBD. Neutralization assays were performed to investigate the neutralization ability of the proteins. The experimental results show that one of the developed proteins is a promising therapeutic approach that will be further improved in the future.