Cyclophilin A‐mediated mitigation of coronavirus SARS‐CoV‐2

Abstract Human cyclophilin A (hCypA) is important for the replication of multiple coronaviruses (CoVs), and cyclosporine A inhibitors can suppress CoVs. The emergence of rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants has sparked concerns that mutations affect the binding ability of the spike (S) protein to the angiotensin‐converting enzyme 2 (ACE2) cell receptor, affecting the severity of coronavirus disease (COVID‐19). Far‐western blotting and surface plasmon resonance (SPR) results revealed that hCypA interacts strongly with the viral SARS‐CoV‐2 receptor‐binding domain (RBD), with a binding affinity of 6.85 × 10−8 M. The molecular interaction between hCypA and the viral protein interface was shown using three‐dimensional structural analysis, which revealed the blocking of key residues on the RBD interface by hCypA. The RBD facilitates binding to the ACE2 receptor. The hCypA–S protein complex suppressed the binding of RBD to the ACE2 receptor, which a required event for CoV entry into the host cell. The reliability of this postulated blocking mechanism of the hCypA–SARS‐CoV2 RBD complex with ACE was confirmed by SPR and molecular interaction lateral flow (MILF) strip assay, which offers the immunochromatographic signal read‐outs. The emergence of new SARS‐CoV‐2 variants with key mutations in RBD had a negligible effect on the binding of the RBD variants to hCypA, indicating an effective mitigation strategy for SARS‐CoV‐2 variants. The MILF strip assay results also highlight the neutralizing effect of hCypA by effectively blocking RBD (wild type and its variants) from binding ACE2. Given the importance of hCypA in viral entry regulation, it has the potential to be used as a target for antiviral therapy.


| INTRODUCTION
An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in December 2019 in Wuhan, China resulted in the new coronavirus (CoV) disease (COVID- 19), triggering a major public health concern worldwide. 1 The World Health Organization (WHO) recognized COVID-19 as a pandemic on March 11, 2020, and the number of infected cases has increased at an alarming rate worldwide. Specific anti-CoV therapies and strategies are critical for the prevention and treatment of COVID-19. Seven different CoVs (SARS-CoV, hCoV-NL63, hCoV-HKU-1, hCoV-OC43, hCoV-229 E, MERS-CoV, and SARS-CoV-2) are currently known to cause respiratory illnesses in humans. 2,3 As a response to the SARS-CoV-2 infection, Paxlovid (Pfizer) was granted emergency use authorization by the United States Food and Drug Administration (FDA). 4,5 It contains the antiviral Nirmatrelvir/ritonavir, which is blocking the activity of SARS-CoV2 main protease (Mpro) and 3CL protease (3CLpro). 6 However, it may have serious side effects and may sometimes be fatal. 7 Also, the appearance of SARS-CoV-2 variants of concerns (VOC) has created an urgent need to develop antiviral agents, new drugs, and vaccines to prevent infection. 8 Cyclophilin (Cyp) proteins play a key role during the lifecycle of viruses from different families, such as human immunodeficiency virus, hepatitis C virus, dengue virus, Japanese encephalitis virus, yellow fever virus, hepatitis B virus, cytomegalovirus, human papillomavirus, influenza A virus, and vesicular stomatitis virus, 9,10 and are also important in the lifecycle of various CoVs. The lifecycles of SARS-CoV, human CoV 229 E (HCoV-229 E) and NL-63 (HCoV-NL63), responsible for mild respiratory infections in humans, and feline infectious peritonitis coronavirus (FPIV), responsible for a fatal disease in cats, are reported to be highly dependent on CypA. [11][12][13][14] Among the different Cyps, CypA is an important protein required for CoV replication and its inhibitor, cyclosporine A (CsA), has the ability to suppress CoV over a broad spectrum. [15][16][17] The 18 kDa human cyclophilin A (hCypA) is an omnipresent protein belonging to the immunophilin family and is conserved and present in both eukaryotes and prokaryotes. hCypA has peptidyl-prolyl cis-trans isomerase (PPIase) activity that catalyzes the cis-trans isomerization of peptide bonds at proline residues and regulates protein folding and trafficking. 10 The CsA molecule can be used to inhibit the binding of hCypA to the SARS-CoV-2 receptor-binding domain (RBD) and to control the hCypA mechanism. 18,19 In addition, the well-known CsA molecule inhibits replication of various viruses by binding to intracellular human cyclophilins, which bind to the SARS-CoV nucleocapsid protein. 16,18 CsA is an important immunosuppressive drug that inhibits PPIase activity by binding to both extracellular and intracellular CypA. 20 It specifically inhibits the protein phosphatase calcineurin (Cn) and prevents the translocation of nuclear factor in activated T cells (NF-AT) from the cytosol to the nucleus, thereby preventing the transcription of pro-inflammatory cytokine encoding genes. 21 While most studies have focused on the intracellular activities of cyclophilins, such as protein folding and molecular chaperone function, 22 studies on the extracellular activity of CypA are limited.

SARS-CoV and MERS-CoV virions carry sufficient quantities of
CypA to maintain their lifecycle and facilitate defects in cell production in their target cells. 15 CypA has also been reported to interact intracellularly with nonstructural SARS-CoV protein 1 (Nsp1). 12 The N protein of SARS-CoV also binds closely to hCypA, and the protein complex formation can be inhibited by CsA, blocking viral replication. 10,14,23 In this context, it is not surprising that CsA, which is a potent hCyp inhibitor with immunosuppressive anti-calcineurin properties, inhibits the in vitro replication of various CoVs, such as HCoV-229 E, HCoV-NL63, FPIV, mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), and SARS-CoV, which are genetically close to SARS-CoV-2. 17,23,24 The homotrimeric spike (S) glycoprotein mediates SARS-CoV-2 entry through the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane. 25 ACE2 receptor recognition by SARS-CoV-2 in humans is similar to that observed in the 2003 SARS-CoV.
The human receptor ACE2 is expressed as a membrane-bound protein present in various organs. 26 At the initial stage of viral replication, binding to the ACE2 receptor is crucial for the entry of SARS-CoV-2 into target cells.
The RBD of S1 includes a core and a receptor-binding motif (RBM), with residues 438-506, that specifically recognizes ACE2. 26 Leu455, Phe456, Ser459, Gln474, Ala475, Phe486, Phe490, Gln493, Pro499, and Asn501 are the key residues in the RBM of the SARS-CoV-2S protein that facilitates the binding of the ACE2 receptor, as has been revealed from the cryo-EM structures of the SARS-CoV-2-hACE2 complex. [27][28][29] RBD is crucial for determining cross-species and human-to-human transmissibility. 28 Antibodies have been used to bind the SARS-CoV-2 RBM in numerous studies, it can be a neutralizer of SARS-CoV-2 and generate information on the nature of immune responses. [30][31][32] To gain insights into the function of hCypA in the SARS-CoV-2 life cycle, we identified and analyzed the interactions of hCypA with S proteins of SARS-CoV-2. These findings also highlight the unique structural characteristics of SARS-CoV-2 RBD, which will help us understand the molecular mechanisms of viral infection.
The emergence of rapidly spreading SARS-CoV-2 variants has sparked concerns about reduced vaccine efficacy. Researchers have found that variants with mutations, which have significant biological functions, have high transmissibility, indicating that key mutations may affect the severity of COVID-19 and viral spread and prevent natural or vaccine-induced immunity. These key mutations significantly affect the binding ability of the S protein to the ACE2 receptor.
In this study, we also analyzed the molecular interactions of the SARS-CoV-2 variants with the hCypA protein to determine the effect of variants on the binding and blocking potential of the hCypA-S protein complex with the ACE2 receptor. In the present study, we affirmed the hCypA interaction with RBD and the interference in binding ACE2-RBD for SARS-CoV-2. A molecular interaction lateral flow (MILF) strip assay was also constructed to determine the inhibitory effect of hCypA on SARS-CoV-2 RBD and its variants. Finally, the hCypA protein-RBD complex suppressed the binding of RBD to the ACE2 receptor, which was a required event for SARS-CoV-2 entry into the host cell. This study provides an important opportunity to determine the efficacy of hCypA as a potential treatment drug target for COVID-19.

| hCypA S protein RBD interactions
The SARS-CoV-2S protein is very similar in sequence (80% sequence identity) and structure (RMSD = 0.411 Å) to the S protein of SARS-CoV (Figures 1a and S1A-C). The CoV intervention strategies aimed at blocking the receptor recognition of SARS-CoV-2S protein can be very useful in restricting the interaction of SARS-CoV-2 with ACE2, thereby preventing virus entry into the target cells. As shown in Fig. S1Ea, the surface plasmon resonance (SPR) binding affinity K D value of 4.48 Â 10 À8 M (48.8 nM) indicates that ACE2 binds marginally strongly to the SARS-CoV-2 S protein relative to SARS-CoV. 27 The binding affinities between ACE2 and SARS-CoV RBDs is the similar binding affinity (K D value of 1.0 Â 10 À8 to 6.0 Â 10 À8 M [10-60 nM]) based on the reported binding results. 33,34 ( Figure S1G).
Structural analysis of SARS-CoV-2 RBD showed that the ACE2 binding mode was almost identical and that most binding residues conserved or shared similar side chain properties (Figures 1a and S1F).
Structural analysis provides a precise target for the binding of hCypA to the SARS-CoV-2 RBD (Figure 1b). To compare the ACE2-interacting residues on the RBD with the hCypA-RBD complex, we employed a structure-guided interaction mapping approach to F I G U R E 1 (a) Structural representation of the S protein RBD (orange) complexed with SARS-CoV-2 receptor ACE2 (light blue). The key residues that take part in the interaction are also shown. (b) Structural representation of hCypA (pink) and S protein RBD (orange) docked complex with interacting region residues labeled on the complex structure. (c) The integrated interaction map of S protein RBD-ACE2 complex and S protein RBD-hCypA complex, highlighting the overlapping regions and residues on RBD.    (Table 1).

To gain insights into the interaction between SARS-CoV-2 RBD
and hCypA, protein-protein interaction analysis was performed. Farwestern blotting detects a target "prey" protein on the membrane using antibody-detectable "bait" protein. 35 Far-western blotting results confirmed the binding of RBD to hCypA and the ACE2 receptor ( Figure 1d). Each protein was loaded on SDS-PAGE, and the RBD was detected using an RBD antibody. The RBD band (control) and ACE2 band signal appeared clearly, indicating that RBD interacts with ACE2 in the PVDF membrane. In addition, the hCypA band signal appears to be smaller than 15 kDa (hCypA protein size 13 kDa), indicating that hCypA interacts with the RBD. Moreover, the SPR results showed that hCypA binds to SARS-CoV-2 RBD with a binding affinity

| hCypA and SARS-CoV-2 variants
To gain molecular insights into the structural difference of hCypA complexed with the SARS-CoV-2 variants, that is, Alpha The Phe486 and Tyr505 residues of RBD on variants that are critical for binding ACE2 also bound hCypA, suggesting an inhibitory effect aided by the hCypA-variant RBD complex on ACE2. SPR analysis was performed to determine the binding ability of the variants to the ACE2 receptor. All seven variants were found to bind strongly to ACE2, with K D values ranging between (10 À8 to 10 À9 M) ( Figure 3 and Table 3). We emphasize that hCypA is able to bind to Alpha, Beta,     appeared on the T line of the AuNP-delta RBD MILF strip, as also indicated by the BSA control ( Figure 4e). Moreover, the band intensity of the T line between wild-type RBD and RBD variants was compared.

| MILF strip assay
As shown in Figure 4f, the band intensity of Delta RBD was higher than that of the other RBD variants and four times stronger than that of the wild-type RBD. The gamma and epsilon variants also showed a weak band intensity and were significantly lower than that of the delta variant. We further verified the activity of SARS-CoV-2 neutralizing antibody with MILF strip assay, FIA, and ELISA ( Figure S3C-F).
We then compared the extracellular neutralization efficiency of the The K D value of binding affinity is shown in Table 3.

| DISCUSSION
The COVID-19 pandemic and its rapid spread worldwide have trig- In our study, protein-protein interactions using bioinformatics tools indicated that hCypA binds to SARS-CoV-2 RBD and that SPR, Note: The graph of SPR affinity analysis is shown in Figure 3 and Figure S2.
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 F I G U R E 4 (a) Illustration of the MILF strip assay for neutralizing antibody test to SARS-CoV-2 and evaluation of the analytical performance of the SARS-CoV-2 MILF strip. (b) Binding interference between ACE2 and SARS-CoV-2 RBD was tested using hCypA. The images and signals of strips exposed to different hCypA concentrations were analyzed by ImageJ software.

| Protein and buffers
The SARS-CoV-2 variants of concern, that is, Alpha (United Kingdom,

| Surface plasmon resonance
The binding affinity of hCypA to RBD proteins and variants was analyzed by SPR using a Biacore X-100 instrument at 25 C. HBS-EP buffer

| Structural analysis
The protein structure of hCypA (Protein Data Bank ID: 3K0M) 44

| MILF strip assay
A MILF strip assay was constructed with four constructs: a sample pad, conjugation pad, nitrocellulose membrane, and absorption pad. Figure S3A shows a schematic diagram of the MILF strip assay. To detect neutralizing antibodies in the blood or serum, AuNPs were conjugated to RBDs. The gold nanoparticles were conjugated to rabbit IgG as a control. The reaction between the AuNPs and the RBDs (or antibodies) was affected by the pH, and effective pH values were identified. First, solutions containing AuNPs were adjusted to a pH of 8.4. Figure 4a shows an illustration of the immunochromatographic test results. The performance of the MILF strip assay was affected by the treatment of the nitrocellulose membrane. Nitrocellulose membranes were blocked with PBS and dried at 37 C for 1 h. Human

DATA AVAILABILITY STATEMENT
All data generated or analyzed for this study are available from the corresponding author upon reasonable request.