The D405N Mutation in the Spike Protein of SARS-CoV-2 Omicron BA.5 Inhibits Spike/Integrins Interaction and Viral Infection of Human Lung Microvascular Endothelial Cells
Abstract:Severe COVID-19 is characterized by angiogenic features, such as intussusceptive angiogenesis, endothelialitis, and activation of procoagulant pathways. This pathological state can be ascribed to a direct SARS-CoV-2 infection of human lung ECs. Recently, we showed the capability of SARS-CoV-2 to infect ACE2-negative primary human lung microvascular endothelial cells (HL-mECs). This occurred through the interaction of an Arg-Gly-Asp (RGD) motif, endowed on the Spike protein at position 403–405, with αvβ3 integr… Show more
“…The coronavirus evolution [ 75 , 76 ], in which new mutants such as omicron variants XBB and XBB1 have emerged [ 77 ], requires investigation of the molecular mechanisms of the transmissibility and pathogenicity. In particular, an important factor for less severe manifestations of COVID-19 disease in the case of mutation D405N of the omicron variant is its impossibility to associate with integrins as possible receptors (different from ACE2) which are capable of binding the S-protein [ 78 ].…”
The association of the S-protein of the SARS-CoV-2 beta coronavirus to ACE2 receptors of the human epithelial cells determines its contagiousness and pathogenicity. We computed the pH-dependent electric potential on the surface of the interacting globular proteins and pH-dependent Gibbs free energy at the association of the wild-type strain and the omicron variant. The calculated isoelectric points of the ACE2 receptor (pI 5.4) and the S-protein in trimeric form (pI 7.3, wild type), (pI 7.8, omicron variant), experimentally verified by isoelectric focusing, show that at pH 6–7, the S1–ACE2 association is conditioned by electrostatic attraction of the oppositely charged receptor and viral protein. The comparison of the local electrostatic potentials of the omicron variant and the wild-type strain shows that the point mutations alter the electrostatic potential in a relatively small area on the surface of the receptor-binding domain (RBD) of the S1 subunit. The appearance of seven charge-changing point mutations in RBD (equivalent to three additional positive charges) leads to a stronger S1–ACE2 association at pH 5.5 (typical for the respiratory tract) and a weaker one at pH 7.4 (characteristic of the blood plasma); this reveals the reason for the higher contagiousness but lower pathogenicity of the omicron variant in comparison to the wild-type strain.
“…The coronavirus evolution [ 75 , 76 ], in which new mutants such as omicron variants XBB and XBB1 have emerged [ 77 ], requires investigation of the molecular mechanisms of the transmissibility and pathogenicity. In particular, an important factor for less severe manifestations of COVID-19 disease in the case of mutation D405N of the omicron variant is its impossibility to associate with integrins as possible receptors (different from ACE2) which are capable of binding the S-protein [ 78 ].…”
The association of the S-protein of the SARS-CoV-2 beta coronavirus to ACE2 receptors of the human epithelial cells determines its contagiousness and pathogenicity. We computed the pH-dependent electric potential on the surface of the interacting globular proteins and pH-dependent Gibbs free energy at the association of the wild-type strain and the omicron variant. The calculated isoelectric points of the ACE2 receptor (pI 5.4) and the S-protein in trimeric form (pI 7.3, wild type), (pI 7.8, omicron variant), experimentally verified by isoelectric focusing, show that at pH 6–7, the S1–ACE2 association is conditioned by electrostatic attraction of the oppositely charged receptor and viral protein. The comparison of the local electrostatic potentials of the omicron variant and the wild-type strain shows that the point mutations alter the electrostatic potential in a relatively small area on the surface of the receptor-binding domain (RBD) of the S1 subunit. The appearance of seven charge-changing point mutations in RBD (equivalent to three additional positive charges) leads to a stronger S1–ACE2 association at pH 5.5 (typical for the respiratory tract) and a weaker one at pH 7.4 (characteristic of the blood plasma); this reveals the reason for the higher contagiousness but lower pathogenicity of the omicron variant in comparison to the wild-type strain.
“…S371F, in the ACE2-receptor-binding domain as well as the adjacent BA.2 specific T376A change, is reported to bear a significant fitness cost and impaired S infectivity, whilst also reducing processing efficiency and/or incorporation into viral pseudo particles [85]. The D405N mutation showed a dramatic loss of function by inhibiting spike/integrins interactions and consequently the virus ability to infect human lung microvascular endothelial cells, thus providing protection against virus-induced endothelial cell dysfunction [86].…”
We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.
“…1 A–C) [ 7 ]. Although the SARS-CoV-2 spike protein has been demonstrated to primarily interact with integrins αVβ3 [ 8 ] and α5β1 [ 9 ], which are RGD-binding integrins, the D405 N variant has recently been shown to inhibit binding to integrin αvβ3 [ 10 ]. Fig.…”
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