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.

SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants represents a difficult challenge due to the loss of vaccine and natural immunity, and increased transmissibility. These variants contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). To understand the effect of RBD mutations, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited since it ignores the conformational dynamics of the RBD itself. Observing that some variants mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic molecular dynamics simulations to investigate the structural dynamics of wt RBD, and that of three variants (alpha, beta and delta). Our results show that in solution, wt RBD presents two distinct conformations: an 'open' conformation where it is free to bind ACE2;and a 'closed' conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. In the delta variant RBD simulations, the closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative 'reversed' one, with a significantly changed orientation of the RBMridge flanking the RBD. These results support the hypothesis that variants impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

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.

Biochemical characterization of SARS-CoV-2 nsp14/nsp10 complex: a promising target for drug design

The global pandemic prompted by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already caused more than 6 million deaths worldwide, calling for urgent effective therapeutic measures. A deep understanding of the mechanisms involved in viral replication is required. Among the nonstructural proteins (nsps) encoded by SARS-CoV-2 genome, there is the nsp14 ribonuclease, the main object of study in this work. Ribonucleases are key factors in the control of all biological processes, ensuring maturation, degradation, and quality control of all types of RNAs. Nsp14 is a bifunctional protein, holding a 3'- 5' exoribonucleolytic activity (ExoN) in the N-terminal domain, stimulated through the interaction with nsp10, and a C-terminal N7-methyltransferase activity (MTase). Both are critical for the coronavirus life cycle. In this work, we provide a complete biochemical characterization of SARS-CoV-2 nsp14-nsp10, addressing several aspects of the complex for the first time. Moreover, using a homology model, we have identified residues involved in the nsp14-nsp10 interaction that were extensively studied. We have confirmed the SARS-CoV-2 nsp14 dual function and we have shown that both ExoN and MTase activities are functionally independent. We demonstrate that the nsp14 MTase activity is independent of nsp10, contrarily to nsp14 ExoN that is upregulated in the presence of the cofactor. Additionally, our results show that the ExoN motif I has a prominent role on the ribonucleolytic activity of SARS-CoV-2 nsp14, contrasting to what was previously observed in other coronaviruses, which can be related to the pathogenesis of SARS-CoV-2. The knowledge provided in this work can serve as a basis to design effective drugs that target the pinpointed residues in order to disturb the complex assembly and affect the viral replication, ultimately, treating COVID-19 and other CoV infections.

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.