What is the role of cyclophilin A in mitigating SARS-CoV-2 infection?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which originated in Wuhan, China in December 2019, has caused the 2019 novel coronavirus (CoV) disease (COVID-19) and caused a worldwide epidemic. caused a serious public health crisis.

The World Health Organization (WHO) declared COVID-19 a pandemic on March 11, 2020. Specific anti-CoV strategies and therapies are essential for the treatment and prevention of COVID-19.

Background

Seven different CoVs are now known to cause respiratory disease in humans. Paxlovid (Pfizer) has been granted Emergency Use Authorization by the U.S. Food and Drug Administration (FDA) in response to SARS-CoV-2. Paxlovid contains antiviral Nirmatrelvir/Ritonavir that blocks the activity of SARS-CoV-2 3CL protease (3CLpro) and main protease (Mpro). However, it can cause serious side effects. Additionally, the emergence of the SARS-CoV-2 variants (VOCs) of concern has created an urgent need for the development of antivirals, new drugs, and new vaccines to prevent infection.

Cyclophilin (Cyp) proteins are involved in the life cycles of various families of viruses, including hepatitis C virus, human immunodeficiency virus, dengue virus, human papillomavirus, cytomegalovirus, influenza A virus, vesicular stomatitis virus, and Japanese encephalitis virus. plays an important role. CoV. NL-63 (HCoV-NL63), human CoV 229 E (HCoV-229 E), and SARS-CoV, which causes mild respiratory infections in humans, and feline infectious peritonitis coronavirus (FPIV), which causes fatal disease. ) life cycle. Cats are reportedly dependent on CypA, which plays a key role in CoV replication. Cyclosporin A (CsA), an inhibitor of CypA, can provide broad-spectrum suppression of CoV.

The 18 kDa human cyclophilin A (hCypA) belongs to the immunophilin family and is present and conserved in prokaryotes and eukaryotes. The hCypA protein is composed of a peptidyl-prolyl cis-trans isomerase (PPIase) activity, capable of catalyzing cis-trans isomerization of peptide bonds at proline residues as well as regulating protein transport and folding. I can. CsA helps block the binding of hCypA to the SARS-CoV-2 receptor binding domain (RBD). It can inhibit PPIase activity by binding to CypA both extracellularly and intracellularly. It has been observed to inhibit the protein phosphatase calcineurin (Cn) and prevent translocation of nuclear factors in activated T cells (NF-AT). However, studies on the extracellular activity of CypA are less well known.

Previous studies have shown that MERS-CoV and SARS-CoV contain large amounts of CypA to maintain their life cycle and promote cell production defects in target cells. CypA has also been observed to interact intracellularly with the non-structural SARS-CoV protein 1 (Nsp1).Therefore, CsA is in vitro Replication of various CoVs genetically close to SARS-CoV-2, including HCoV-NL63, HCoV-229 E, SARS-CoV, infectious avian bronchitis virus (IBV), murine hepatitis virus (MHV), and FPIV.

homotrimeric spike (S) glycoprotein Mediates SARS-CoV-2 entry through host angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 ACE2 receptor recognition has been observed to be similar to his SARS-CoV in 2003. Human ACE2 receptor expression can be observed as a membrane-bound protein in various organs. The S1 RBD contains a core and a receptor binding motif (RBM) that precisely recognizes ACE2. In addition, RBD is important for determining human-to-human and transspecies transmissibility. Furthermore, to understand the function of hCypA in the life cycle of SARS-CoV-2, the identification and analysis of the interaction between hCypA and S protein of SARS-CoV-2 was performed.

The emergence of SARS-CoV-2 variants has raised concerns. efficacy of vaccination. Researchers show that variants lead to high contagiousness, affect the severity of COVID-19, and include mutations that may interfere with vaccines and innately induced immunity. These mutations may also affect the binding of S protein to her ACE2 receptor.

new research in Bioengineering and Translational Medicine The journal intends to analyze the molecular interactions between the hCypA protein and SARS-CoV-2 variants to determine the effects of the variants on the blocking and binding potential of the hCypA–S protein complex with the ACE2 receptor. It was intended.

About research

This study involved purchasing anti-SARS-CoV-2 along with various SARS-CoV-2 variants including alpha, beta, delta, gamma, omicron, kappa, epsilon, deltochron, and lambda variants. Neutralizing antibody, human cyclophilin A, recombinant ACE2, anti-rabbit IgG, and anti-human IgG antibodies. Far-Western blotting was then performed using purified ACE2, hCypA and RBD. Surface plasma resonance was used to determine the binding affinity of hCypA to RBD proteins and variants.

To analyze the influence of hCypA on S-protein ACE2 interaction, structural analysis of the SARS-CoV-2–hCypA complex docked with ACE2 was performed. Finally, we constructed a molecular interaction lateral flow (MILF) assay to obtain a readout of the immunochromatographic signal.

Survey results

The results showed that the SARS-CoV-2 S protein is highly similar to the SARS-CoV S protein in structure and sequence. We also observed that the binding affinities between ACE2 and SARS-CoV RBD were similar. The hCypA protein was observed to engulf her RBM of SARS-CoV-2, blocking access to key residues involved in interaction with ACE2. Furthermore, the active site of hCypA was observed to consist of seven residues that interact with CsA and are involved in PPIase activity. The hCypA-RBD complex interface is reported to be stabilized by many interactions and five intermolecular hydrogen bonds.

Binding of RBD to hCypA and ACE2 receptors was confirmed through Far-Western blotting results. High binding energy and binding affinity values ​​were observed when hCypA interacted with the SARS-CoV-2 RBD. The binding of ACE2 to RBD was observed to decrease in the presence of hCypA. Furthermore, we observed that the RBD interaction was reduced in the presence of the hCypA-CsA complex. We observed that the binding affinity of the hCypA-CsA complex was higher compared to that of the hCypA-RBD complex. Therefore, binding of hCypA–CsA favors the binding of ACE2 to her RBD.

Except for Delta, no significant conformational changes were observed in SARS-CoV-2 variants compared to the wild-type RBD-hCypA complex. hCypA was reported to bind to her RBD of all variants except Delta. Positive residues in Delta RBD have been reported to cause steric hindrance during hCypA binding. The MILF strip assay results also confirmed that hCypA binding inhibited the interaction of her RBD with ACE2. Similar results were obtained with all SARS-CoV-2 variants except Delta.

Therefore, the current study demonstrated that hCypA plays an important role in regulating SARS-CoV-2 within the host. It can bind to RBD and prevent interaction with host ACE2, suggesting that hCypA can be used as a potential target for antiviral therapy.