Human Host Protein Cyclophilin-A Plays A Role In Aiding SARS-CoV-2 infections

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South Korean researchers from Chungbuk National University, Shin Ansan University and Jeonbuk National University have found that the human host protein Cyclophilin-A plays a role in in aiding SARS-CoV-2 infections.

Cyclophilins (CYPs) are a family of proteins named after their ability to bind to ciclosporin (cyclosporin A), and function as immunosuppressants. They are found in all domains of life. These proteins have peptidyl prolyl isomerase activity, which catalyzes the isomerization of peptide bonds from trans form to cis form at proline residues and thus facilitates protein folding as well.

The study findings were published in the peer reviewed journal: Bioengineering & Translational Medicine.
https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10436

The COVID-19 pandemic and its rapid spread worldwide have triggered a global health emergency. Numerous therapeutic approaches have been proposed, and the recognition of cell receptors by CoVs is critical for the determination of viral infection, pathogenesis, and host range.

The SARS-CoV-2S protein is the main protein used as a target in COVID-19 vaccines. The RBD of the S protein binds to the ACE2 receptor on host cells and initiates virus-host cell membrane fusion, which is crucial for viral infection.

As a result, screening inhibitors that inhibit RBD–ACE2 interaction are critical for the treatment of COVID-19.

One of the most effective strategies to inhibit viral entry is targeting host or virus-related components by directly blocking or indirectly interfering with the interaction between RBD and human ACE2. The RBD, as the key region for binding receptors, attracts antibodies and proteins targeting the conserved residues and offers great potential for the development of potent cross-reactive therapeutic agents against various CoVs.

The binding affinity described in this study showed that ACE2 bound marginally strongly to the SARS-CoV-2S protein relative to SARS-CoV, as they have high structural similarity, coinciding with similar findings reported by other groups.27, 33, 40

SARS-CoV-2 binding to ACE2 is dominated by the RBD/ACE2 interface and blocking the interacting residues of RBD can inhibit specific binding to the ACE2 receptor. The protein hCypA plays a critical role in the life cycle of many CoVs (HCoV-229 E, HCoV-NL63, FPIV, SARS-CoV, and MERS-CoV).

In our study, protein–protein interactions using bioinformatics tools indicated that hCypA binds to SARS-CoV-2 RBD and that SPR, far-western blot, and MILF strip assays revealed their binding interactions. Protein structure prediction and protein–protein interaction analysis using bioinformatics tools can help screen new drugs against other variants or diseases.

We observed that extracellular hCypA binds SARS-CoV-2 RBD at the key RBM residue interface involved in interactions with the ACE2 receptor and overlaps with the ACE2 binding region. This hinders virus interactions, thereby mitigating its activity and restricting the entry of SARS-CoV-2 into the host cell.

A strong interaction between the hCypA–RBD complex, as demonstrated by SPR and structural analysis, decreased the RBD-ACE2 binding capacity, working as a masking mechanism to reduce RBD exposure to the ACE2 receptor. The hCypA acts as a potential inhibitor that can efficiently block SARS-CoV-2 binding.

The CsA molecule can be used as an immune-suppressor to inhibit the binding of hCypA to the SARS-CoV-2 RBD and to control the hCypA mechanism. In addition, the well-known CsA molecule inhibits the replication of various viruses by binding to intracellular human cyclophilins, which bind to the SARS-CoV nucleocapsid protein.16, 41

Although hCypA can inhibit SARS-CoV-2 binding to host cells, even if the virus penetrates into the cell, it can interrupt viral replication by inhibiting the nucleocapsid–hCypA complex through CsA in the latent stage of viral infection.42, 43 The emergence of variants with mutations in the S protein affects the binding ability of the virus to the ACE2 receptor and exhibits high transmissibility and faster spreading.

SPR results showed that the variants bind tightly to hCypA, suggesting no significant alterations to the complex structure due to mutations in the residues in the variants. The hCypA protein overlaps the RBM on all the variants and prevents the protein from tightly binding to the ACE2 receptor, except for the delta variant.

Therefore, the delta variant evades the neutralizing effect of hCypA, as the T478K mutation on the RBD causes steric hindrance due to the surface potential shift from neutral to positive, which reduces stability and forces the flexible loop region on hCypA to shift away from the RBM region.

The ACE2 receptor easily binds to the hCypA–delta RBD complex in open RBM regions, resulting in swift virus entry into the host cell. The N501Y mutation on RBD increases binding to ACE2 receptor; however, the combination of Q498R with N501Y in the omicron variant is suspected to further increase the binding affinity with ACE2.

The strong binding of hCypA on the mutated RBD residue interface on omicron variant is expected to not only prevent its spread but also neutralize its transmissibility. The hCypA–RBD interaction highlights a new strategy for preventing a possible SARS-CoV-2 infection pathway against host cells and serves as a feasible approach for preventing SARS-CoV-2 infection.

The MILF strip assay results also confirmed the binding mechanism of hCypA with RBD and its variants, where hCypA inhibits the binding of variants to the ACE2 receptor, except for the delta variant.

The hCypA protein binds to the RBD of SARS-CoV-2 with a high affinity and possesses neutralizing ability. We visually confirmed the protein–protein binding interactions using the MILF strip assay.

It can be used as a tool for evaluating the protein–protein interactions and molecular binding forces.

Furthermore, MILF strip can be used to determine the inhibitory effect of hCypA on SARS-CoV-2 RBD and its variants.

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