What is the role of cyclophilin-A in alleviating SARS-CoV-2 infections?

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan, China, in December 2019 led to the novel coronavirus (CoV) disease 2019 (COVID-19), causing a significant worldwide public health crisis.

The World Health Organization (WHO) declared the COVID-19 pandemic on 11th March 2020 due to an alarming increase in cases worldwide. Specific anti-CoV strategies and therapies are essential for the treatment and prevention of COVID-19.

Background

Currently, seven different CoVs are known to cause respiratory illnesses in humans. 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 protease (3CLpro) and main protease (Mpro). However, it can lead to serious side effects. Moreover, the emergence of SARS-CoV-2 variants of concern (VOC) has led to the urgent requirement for developing antiviral agents, new drugs, and new vaccines to prevent infection.

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. 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.

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. 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.

Previous studies have 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.

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 includes comprises a core and a  receptor-binding motif (RBM) that accurately recognizes ACE2. Additionally, 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 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.

A new study in the Bioengineering and Translational Medicine journal aimed to analyze 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.

About the study

The study involved the purchase 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. After that far-western blotting was carried out using purified ACE2, hCypA, and RBD. Surface plasma resonance was used to determine the binding affinity of hCypA to RBD proteins and variants.

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.

Study findings

The results 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. Additionally, 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 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. Moreover, RBD interactions were 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.

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.

Therefore, the current study 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.

Written by

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.