SARS-CoV-2 News: South Korean Researchers Discover That Human Host Protein Cyclophilin-A Plays A Role In In Aiding SARS-CoV-2 infections!

SARS-CoV-2 News: 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.
 
It has been found that 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 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.
 
For the first time, the molecular interaction between hCypA and the viral protein interface was revealed 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 is 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.
 
Interestingly, 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.
 
The study findings were published in the peer reviewed journal: Bioengineering & Translational Medicine.
https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10436
 
At present, seven different CoVs are known to cause respiratory illnesses in humans.
 
According to various SARS-CoV-2 News coverages, the drug Paxlovid (Pfizer) was approved for emergency use authorization by the United States Food and Drug Administration (FDA) in response to SARS-CoV-2. Paxlovid contains the antiviral Nirmatrelvir/Ritonavir that blocks the activity of the SARS-CoV-2 3CL pr otease (3CLpro) and main protease (Mpro). However, the drug can also lead to serious side effects. The other U.S. FDA approved antivirals such as remdesivir and also molnupiravir have been found to cause certain hepatic, nephrotic an also cardio toxicity issues as well their efficacy is questionable.
 
The emergence of SARS-CoV-2 variants of concern (VOC) and various new Omicron sub-lineages along with a variety of recombinant variants has led to the urgent need for developing new antiviral agents, new drugs, and new vaccines to prevent infection.
 
It has been already known that Cyclophilin (Cyp) proteins play an important role during the lifecycle of viruses from different families, such as hepatitis C virus, human immunodeficiency virus, dengue virus, human papillomavirus, cytomegalovirus, influenza A virus, vesicular stomatitis virus, Japanese encephalitis virus, and various CoVs.
 
Interestingly, the lifecycles of NL-63 (HCoV-NL63), human CoV 229 E (HCoV-229 E), and SARS-CoV that causes mild respiratory infections in humans, as well as feline infectious peritonitis coronavirus (FPIV) that causes fatal disease in cats, is reported to be dependent on CypA, which plays an important role in CoV replication. Cyclosporine A (CsA), which is the inhibitor of CypA can provide a broad-spectrum suppression of CoV.
 
It is well established that the 18 kDa human cyclophilin A (hCypA) belongs to the immunophilin family and is both present and conserved in prokaryotes and eukaryotes.
 
The hCypA protein consists of peptidyl-prolyl cis-trans isomerase (PPIase) activity and can carry out the catalysis of cis-trans isomerization of peptide bonds at proline residues as well as regulate protein trafficking and folding.
 
Cyclosporine A or CsA can be useful in inhibiting the binding of hCypA to the SARS-CoV-2 receptor-binding domain (RBD). It can inhibit PPIase activity by binding CypA both extracellularly and intracellularly. It has been observed to inhibit the protein phosphatase calcineurin (Cn) and prevent the translocation of a nuclear factor in activated T cells (NF-AT) which in turn prevents the transcription of genes encoding the pro-inflammatory cytokines. However, studies on the extracellular activity of CypA are not well known.
 
Past research has indicated that MERS-CoV and SARS-CoV contain significant amounts of CypA for maintaining their lifecycle and expediting any defects in cell production in their target cells. CypA has also been observed to interact intracellularly with non-structural SARS-CoV protein 1 (Nsp1). Therefore, CsA can inhibit the in vitro replication of various CoVs, including HCoV-NL63, HCoV-229 E, SARS-CoV, avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), and FPIV, which are genetically close to SARS-CoV-2.
 
It has already well known that the homotrimeric spike (S) glycoprotein mediates the entry of SARS-CoV-2 through the host the angiotensin-converting enzyme 2 (ACE2) receptor. SARS-CoV-2 ACE2 receptor recognition is observed to be similar to the 2003 SARS-CoV. The expression of the human ACE2 receptor can be observed as a membrane-bound protein in various organs. The RBD of S1 comprises a core and a receptor-binding motif (RBM) that accurately recognizes ACE2.
 
Also, RBD is important for determining human-to-human and cross-species transmissibility. Furthermore, identification and analysis of interactions between hCypA and S proteins of SARS-CoV-2 have been carried out to understand the function of hCypA in the life cycle of SARS-CoV-2.
 
The rapid emergence of SARS-CoV-2 variants has increased concerns about the efficacy of vaccines. Researchers have indicated that the variants comprise mutations that can lead to high transmissibility, affect COVID-19 severity, and prevent vaccine and natural-induced immunity. These mutations can also affect the binding of the S protein to the ACE2 receptor.
 
This study is the first to have analyzed the molecular interactions between the hCypA protein and the SARS-CoV-2 variants to determine the impact of variants on the blocking and binding potential of the hCypA–S protein complex with the ACE2 receptor.
 
The research involved the procurement of different SARS-CoV-2 variants that included, Alpha, Beta, Delta, Gamma, Omicron, Kappa, Epsilon, Deltcron, and Lambda variants along with anti-SARS-CoV-2 neutralizing antibodies, human cyclophilin A, recombinant ACE2, anti-rabbit IgG, and anti-Human IgG antibody.
 
The study team carried out far-western blotting using purified ACE2, hCypA, and RBD. Surface plasma resonance was used to determine the binding affinity of hCypA to RBD proteins and variants.


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. (c) The calibration graph as control line (C line) and test line (T line, ACE2 zone) ratio of hCypA concentration. (d) MILF strip assay that highlights the neutralizing ability of hCypA was confirmed at the T line using AuNP-variant RBDs. MILF strip assay test results of the hCypA with the AuNP-variants RBDs (e), and the calibration graph as the T/C ratio (f).

Detailed structural analysis of SARS-CoV-2–hCypA complexes docked with ACE2 was carried out to analyze the impact of hCypA on S protein ACE2 interactions. Finally, a molecular interaction lateral flow (MILF) assay was constructed to obtain immunochromatographic signal read-outs.
 
The study findings indicated that the SARS-CoV-2 S protein was very similar in structure and sequence to the SARS-CoV S protein. Also, the binding affinity between ACE2 and SARS-CoV RBDs was observed to be similar. The hCypA protein was observed to engulf the RBM of SARS-CoV-2 and block access to key residues that are involved in the interaction with ACE2.
 
The hCypA’s active site was observed to consist of seven residues that interact with CsA and are involved in PPIase activity. The hCypA–RBD complex interface was reported to be stabilized by many interactions and five intermolecular hydrogen bonds.
 
The actual binding of RBD to hCypA and the ACE2 receptor was confirmed through the far-western blotting results. High binding energy and binding affinity values were observed when hCypA interacted with SARS-CoV-2 RBD. The binding of ACE2 with RBD was observed to be reduced in the presence of hCypA.
 
RBD interactions were also observed to be reduced in the presence of the hCypA–CsA complex. The binding affinity of the hCypA–CsA complex was observed to be higher as compared to the binding affinity of the hCypA–RBD complex. Therefore, the binding of hCypA–CsA favors the binding of ACE2 to RBD.
 
However, no significant structural alterations were observed for the SARS-CoV-2 variants as compared to the wild-type RBD–hCypA complex, except for Delta. hCypA was reported to bind to the RBD of all the variants except Delta. The positive residues on the Delta RBD were reported to cause steric hindrance during the binding of hCypA. The results of the MILF strip assay also confirmed that the binding of hCypA inhibited the interaction of RBD with ACE2. Similar results were obtained for all SARS-CoV-2 variants except Delta.
 
The study findings demonstrated that hCypA plays an important role in regulating SARS-CoV-2 inside the host. It can bind to RBD and prevent its interaction with host ACE2, suggesting that hCypA can be used as a potential target for antiviral therapy.
 
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