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 of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs 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). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs 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 (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (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. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a 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 RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics. One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance. In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3-to-5 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs. Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3-5 exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN. Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV. We have studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function contrasting to the functionality of these conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronavirus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2. The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.