Biochemical Characterization of SARS‐CoV‐2 Spike RBD Mutations and Their Impact on ACE2 Receptor Binding

Infection of mammalian cells by SARS‐CoV‐2 coronavirus requires primary interaction between the receptor binding domain (RBD) of viral Spike protein and the host cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein. Several mutations in the RBD of SARS‐CoV‐2 spike protein have been reported for several variants and resulted in wide spread of the COVID pandemic. For instance, the double mutations L452R and E484Q present in the Indian B.1.617 variant have been suggested to cause evasion of host immune response. The common RBD mutations N501Y and E484K were found to enhance the interaction with ACE2 receptor. In the current study, we analyzed the biosynthesis and secretion of the RBD double mutant L452R and E484Q in comparison to the wild‐type RBD and the individual mutations N501 and E484K in mammalian cells. Moreover, we evaluated the interaction of those variants with ACE2 by means of expression of the S protein and co‐immunoprecipitation with ACE2. Our results revealed that the double RBD mutations L452R and E484Q resulted in a higher expression level and secretion of Spike S1 protein as compared with other mutations. In addition, an increased interaction of these mutant forms with ACE2 in Calu3 cells was observed. Altogether, our findings highlight the impact of continuous S1 mutations on the pathogenicity of SARS‐CoV‐2 and provides further biochemical evidence for the dominance and high transmissibility of the double indian mutations

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Biochemical Characterization of SARS-CoV-2 Spike RBD Mutations and Their Impact on ACE2 Receptor Binding.

Infection of mammalian cells by SARS-CoV-2 coronavirus requires primary interaction between the receptor binding domain (RBD) of the viral spike protein and the host cell surface receptor angiotensin-converting enzyme 2 (ACE2) glycoprotein. Several mutations in the RBD of SARS-CoV-2 spike protein have been reported for several variants and resulted in wide spread of the COVID pandemic. For instance, the double mutations L452R and E484Q present in the Indian B.1.617 variant have been suggested to cause evasion of the host immune response. The common RBD mutations N501Y and E484K were found to enhance the interaction with the ACE2 receptor. In the current study, we analyzed the biosynthesis and secretion of the RBD double mutants L452R and E484Q in comparison to the wild-type RBD and the individual mutations N501 and E484K in mammalian cells. Moreover, we evaluated the interaction of these variants with ACE2 by means of expression of the S protein and co-immunoprecipitation with ACE2. Our results revealed that the double RBD mutations L452R and E484Q resulted in a higher expression level and secretion of spike S1 protein than other mutations. In addition, an increased interaction of these mutant forms with ACE2 in Calu3 cells was observed. Altogether, our findings highlight the impact of continuous S1 mutations on the pathogenicity of SARS-CoV-2 and provide further biochemical evidence for the dominance and high transmissibility of the double Indian mutations.

Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) causes a severe respiratory disease in humans. The MERS-CoV spike (S) glycoprotein mediates viral entry into target cells. For this, MERS-CoV S engages the host cell protein dipeptidyl peptidase 4 (DPP4, CD26) and the interface between MERS-CoV S and DPP4 has been resolved on the atomic level. Here, we asked whether naturally-occurring polymorphisms in DPP4, that alter amino acid residues required for MERS-CoV S binding, influence cellular entry of MERS-CoV. By screening of public databases, we identified fourteen such polymorphisms. Introduction of the respective mutations into DPP4 revealed that all except one (Δ346-348) were compatible with robust DPP4 expression. Four polymorphisms (K267E, K267N, A291P and Δ346-348) strongly reduced binding of MERS-CoV S to DPP4 and S protein-driven host cell entry, as determined using soluble S protein and S protein bearing rhabdoviral vectors, respectively. Two polymorphisms (K267E and A291P) were analyzed in the context of authentic MERS-CoV and were found to attenuate viral replication. Collectively, we identified naturally-occurring polymorphisms in DPP4 that negatively impact cellular entry of MERS-CoV and might thus modulate MERS development in infected patients.