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Structural evolution of severe acute respiratory syndrome coronavirus 2: Implications for adhesivity to angiotensin-converting enzyme 2 receptors and vaccines

Published:August 11, 2022DOI:https://doi.org/10.1016/j.ejim.2022.08.012

      Highlights

      • The Omicron BA.5 variant currently accounts for the majority of cases of SARS-CoV-2 infection.
      • ACE2 receptors mediate the entry of SARs-CoV-2 into cells.
      • The mutated spike protein of Omicron BA.5 shows a markedly closer binding to ACE2.
      • The process of internalization of ACE2 receptors which accompanies their binding with the viral and vaccine spike proteins eliminates enzymatic activity of ACE2 receptors on the outer cell surface, which degrades Angiotensin II into Angiotensin1–7.
      • The spike proteins generated by new vaccines, which are built on the sequence of Omicron BA.5, are thus expected to exert a closer binding to ACE2 receptors when compared to first-generation vaccines, with potential enhancement of Angiotensin II activity versus Angiotensin1–7.

      Keywords

      Abbreviations:

      SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), S (spike), ACE2 (angiotensin-converting enzyme 2), Ang (sngiotensin), RAS (renin-angiotensin system), COVID-19 (coronavirus disease 2019), RBD (receptor binding domain), ORF (open reading frame), Kd (dissociation constant)
      The Omicron BA.5 variant currently accounts for the majority of cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (https://covid.cdc.gov/covid-data-tracker/#variant-proportions). This subvariant evolved from the Omicron lineage and became more contagious. Furthermore, data suggest that Omicron sublineages may evade polyclonal neutralizing antibody responses elicited by primary vaccine series [
      • Bowen J.E.
      • Addetia A.
      • Dang H.V.
      • Stewart C.
      • Brown J.T.
      • Sharkey W.K.
      • Sprouse K.R.
      • Walls A.C.
      • Mazzitelli I.G.
      • Logue J.K.
      • Franko N.M.
      • Czudnochowski N.
      • Powell A.E.
      • Dellota Jr., E.
      • Ahmed K.
      • Ansari A.S.
      • Cameroni E.
      • Gori A.
      • Bandera A.
      • Posavad C.M.
      • Dan J.M.
      • Zhang Z.
      • Weiskopf D.
      • Sette A.
      • Crotty S.
      • Iqbal N.T.
      • Corti D.
      • Geffner J.
      • Snell G.
      • Grifantini R.
      • Chu H.Y.
      • Veesler D
      Omicron spike function and neutralizing activity elicited by a comprehensive panel of vaccines.
      ,
      • Wang Q.
      • Guo Y.
      • Iketani S.
      • Nair M.S.
      • Li Z.
      • Mohri H.
      • Wang M.
      • Yu J.
      • Bowen A.D.
      • Chang J.Y.
      • Shah J.G.
      • Nguyen N.
      • Chen Z.
      • Meyers K.
      • Yin M.T.
      • Sobieszczyk M.E.
      • Sheng Z.
      • Huang Y.
      • Liu L.
      • Ho D.D.
      Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BA.5.
      ]. Of note, development of antibodies to SARS-CoV-2 largely revolves around the viral spike (S) glycoprotein [
      • Almehdi A.M.
      • Khoder G.
      • Alchakee A.S.
      • Alsayyid A.T.
      • Sarg N.H.
      • Soliman S.S.M.
      SARS-CoV-2 spike protein: pathogenesis, vaccines, and potential therapies.
      ], which mediates host cell entry by binding to a cell surface receptor, angiotensin-converting enzyme 2 (ACE2, Fig. 1) [
      • Verdecchia P.
      • Reboldi G.
      • Cavallini C.
      • Mazzotta G.
      • Angeli F.
      [ACE-inhibitors, angiotensin receptor blockers and severe acute respiratory syndrome caused by coronavirus].
      ]. ACE2 receptors are composed by 805 amino acids and use a single extracellular catalytic domain to cleave an amino acid from angiotensin (Ang) I to form Ang1,9 and to remove an amino acid from Ang II to form Ang1–7[
      • Verdecchia P.
      • Reboldi G.
      • Cavallini C.
      • Mazzotta G.
      • Angeli F.
      [ACE-inhibitors, angiotensin receptor blockers and severe acute respiratory syndrome caused by coronavirus].
      ]. The process of internalization of ACE2 receptors which accompanies the link with the viral S protein, eliminates the enzymatic activity of ACE2 receptors on the outer cell surface. This phenomenon could dysregulate the renin-angiotensin system (RAS), with reduced final generation of Ang1,7 from Ang II [
      • Ramos S.G.
      • Rattis B.
      • Ottaviani G.
      • Celes M.R.N.
      • Dias E.P.
      ACE2 down-regulation may act as a transient molecular disease causing RAAS dysregulation and tissue damage in the microcirculatory environment among COVID-19 patients.
      ,
      • Angeli F.
      • Reboldi G.
      • Verdecchia P.
      SARS-CoV-2 infection and ACE2 inhibition.
      ]. Since Ang1,7 exert anti-inflammatory, antithrombotic and vasodilating effects, the ascendency of Ang II over Ang1–7 might contribute to trigger inflammation, thrombosis and vasoconstriction [
      • Angeli F.
      • Zappa M.
      • Reboldi G.
      • Trapasso M.
      • Cavallini C.
      • Spanevello A.
      • Verdecchia P.
      The pivotal link between ACE2 deficiency and SARS-CoV-2 infection: one year later.
      ,
      • Verdecchia P.
      • Cavallini C.
      • Spanevello A.
      • Angeli F.
      The pivotal link between ACE2 deficiency and SARS-CoV-2 infection.
      ]. Several investigations demonstrated the development of an “Ang II storm” or “Ang II intoxication” during the acute phase of infection[
      • Angeli F.
      • Reboldi G.
      • Trapasso M.
      • Zappa M.
      • Spanevello A.
      • Verdecchia P.
      COVID-19, vaccines and deficiency of ACE2 and other angiotensinases. Closing the loop on the "Spike effect".
      ]. Wu and co-workers demonstrated a significant increase in Ang II levels among coronavirus disease 2019 (COVID-19) patients [
      • Wu Z.
      • Hu R.
      • Zhang C.
      • Ren W.
      • Yu A.
      • Zhou X
      Elevation of plasma angiotensin II level is a potential pathogenesis for the critically ill COVID-19 patients.
      ]. More specifically, they found increased Ang II levels in 90.2% of COVID-19 patients, and a direct association between plasma Ang II levels and COVID-19 severity [
      • Wu Z.
      • Hu R.
      • Zhang C.
      • Ren W.
      • Yu A.
      • Zhou X
      Elevation of plasma angiotensin II level is a potential pathogenesis for the critically ill COVID-19 patients.
      ]. Similar results were obtained in a clinical study investigating disease severity in SARS-CoV-2 infected patients: Liu and co-workers found that Ang II levels in the plasma samples were significantly increased and linearly associated with viral load and lung damage [
      • Liu Y.
      • Yang Y.
      • Zhang C.
      • Huang F.
      • Wang F.
      • Yuan J.
      • Wang Z.
      • Li J.
      • Li J.
      • Feng C.
      • Zhang Z.
      • Wang L.
      • Peng L.
      • Chen L.
      • Qin Y.
      • Zhao D.
      • Tan S.
      • Yin L.
      • Xu J.
      • Zhou C.
      • Jiang C.
      • Liu L.
      Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury.
      ].
      Fig 1
      Fig. 1Structure and interaction of SARS-CoV-2 spike glycoprotein with ACE2 receptor (see text for details).
      We evaluated the effect of mutations of BA.5 subvariant on the receptor binding domain (RBD) of the S protein, the overall conformational dynamics of the S protein and its adhesivity to ACE2 receptors. We downloaded the proteins sequence and structure for SARS-CoV-2 S protein and ACE2 receptor from the Protein Data Bank [
      • ww PDBc.
      Protein Data Bank: the single global archive for 3D macromolecular structure data.
      ]. We analyzed the nucleotide sequence of BNT162b2 vaccine, accession MQ287666 (Patent WO2021214204). We annotated the open reading frame (ORF) coding for vaccine S and we translated into protein sequence with Translate tool (https://web.expasy.org/translate). The Pymol mutagenesis wizard (https://pymol.org) was used to introduce the specific mutations at the appropriate residues in wild type SARS-CoV-2 S in order to create Alpha B.1.1.7 and Omicron BA.5 variants. The online server EMBOSS Pepstats (https://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/) was used to calculate the amino acid composition, molecular weight, distribution of charged residues, hydropathicity, aliphatic index and instability index (for both Alpha B.1.1.7 and Omicron BA.5 subvariants). In order to verify Pepstats results, we conducted a second analysis with ProtParam [
      • Gasteiger E.
      • Hoogland C.
      • Gattiker A.
      • Duvaud S.
      • Wilkins M.R.
      • Appel R.D.
      • Bairoch A.
      Protein identification and analysis tools on the ExPASy Server.
      ] and AA‑prop (http://www.biogem.org/tool/aa-prop/). In addition, in order to compare and visualize residue-level physicochemical properties and to assess the impact of mutations on function we adopted PROVEAN (http://provean.jcvi.org/index.php) and VOLPES (http://volpes.univie.ac.at/) software, respectively. To evaluate binding affinity (ΔG) and Kd prediction we used the PRODIGY webserver (https://wenmr.science.uu.nl/prodigy/). After preparing the protein, Cluspro (https://cluspro.bu.edu/login.php) was used to dock the reference Alpha and Omicron to the ACE2 receptor. Finally, we generate the 3D figure with Pymol graphical software (https://pymol.org/2/) and we did a pairwise alignment of the RBD's sequence of BNT162b2 vaccine, alpha and omicron variants with Clustal omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to evaluate the possible sequence of new vaccine and their possible effects,
      We assessed the impact of mutations on the function of S protein and computed the binding affinity (ΔG) and dissociation constant (Kd) (Fig. 2, upper panel). We found a 63-fold increase in binding affinity with ACE2 of Omicron BA.5 S protein, when compared with Alpha B.1.1.7 variant (Fig. 2, middle panel). Fig. 2 shows an example of a new bond with the ACE2 receptor resulting from the Q498R mutation in Omicron BA.5. Moreover, mutations in the Omicron BA.5 S protein have made it more positively charged than the Alpha variant's spike. This change in charge may attracts Omicron BA.5 toward the negatively charged ACE2 protein even across relatively large distances (Figure, middle panel).
      Fig 2
      Fig. 2Mutations observed in Omicron BA.5 variant (and comparison with Alpha B1.1.7) with 3D structure of S protein (upper Panel). Affinity of both Alpha B.1.1.7 and Omicron BA.5 to ACE2 (middle Panel) and comparison alignment of the receptor binding domain's sequence of BNT162b2 vaccine and the two variants (lower Panel) are also reported.
      The pairwise alignment of the RBD's sequence of BNT162b2 vaccine revealed an identical sequence with Alpha B.1.1.7 variant (Fig. 2, lower panel). For effect of mutations, the aminoacidic sequences of the RBD markedly differed between Alpha B.1.1.7 and Omicron BA.5 with significant functional changes (Fig. 2, lower panel).
      In conclusion, the mutated S protein of Omicron BA.5 showed a 63-fold higher adhesivity to ACE2 receptors, with expected enhanced activity of Ang II and concomitant Ang1–7 deficiency. Finally, the higher adhesivity to ACE2 receptors by the S protein generated by new vaccines eventually built on the aminoacidic sequence of Omicron BA.5 might enhance the imbalance between Ang II overactivity and of Ang1–7 deficiency.
      Basic and clinical research is urgently needed to investigate the clinical impact of dysregulated RAS axis on SARS-CoV-2 disease and vaccination. Moreover, the potentially detrimental impact of the interactions between S proteins (viral or vaccine-induced) and ACE2 and other angiotensinases (involved in the processing of Ang II to Ang1,7) remains to be determined [
      • Angeli F.
      • Reboldi G.
      • Trapasso M.
      • Zappa M.
      • Spanevello A.
      • Verdecchia P.
      COVID-19, vaccines and deficiency of ACE2 and other angiotensinases. Closing the loop on the "Spike effect".
      ]. New experimental and clinical data exploring the relationships between different mechanisms of Ang II cleavage and accumulation will be valuable in guiding the development of vaccines and other therapeutic strategies against SARS-CoV-2 pandemic [
      • Angeli F.
      • Reboldi G.
      • Ageing Verdecchia P.
      ACE2 deficiency and bad outcome in COVID-19.
      ,
      • Angeli F.
      • Spanevello A.
      • Reboldi G.
      • Visca D.
      • Verdecchia P.
      SARS-CoV-2 vaccines: lights and shadows.
      ].

      Declaration of Competing Interest

      None.

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