Genetic deficiency and polymorphisms of cyclophilin A reveal its essential role for Human Coronavirus 229E replication

Abstract

Replication of coronaviruses is inhibited in vitro by cyclosporin A, a well-known immunosuppressive drug which binds to cellular cyclophilins thus inactivating their enzymatic cis-trans peptidyl-prolyl isomerase function. Latter is required for proper folding of cellular proteins and of proteins of several viruses. Here, we summarize present knowledge on the role of cyclophilin A during coronavirus replication. We present data on the effect of cyclophilin A single nucleotide polymorphism mutants on the replication of human CoV-229E demonstrating the requirement of proper cyclophilin A function for virus propagation. Results define cellular cyclophilin A as a host target for inhibition of coronaviruses ranging from relatively mild common cold to highly pathogenic SARS-CoV and MERS-CoV viruses with the perspective of disclosing non-immunosuppressive cyclosporin A analogs to broadly inactivate the coronavirus family.

Figure 1

Crystal structure of human CypA complexed with CsA (1CWA, pdb database, modified) and with coding non-synonymous PPIA gene SNPs. SNPs with accompanying amino acid exchanges introduced in Huh-7.5 PPIA manipulated cell lines [46] are Rs61747111 (D66E), rs17850033 (I89T), rs1059983 (E84D), rs11547706 (G96D), rs17850166 (N106I), rs9769523 (E134K). CypA is shown as a β-sheet structure with the SNP amino acids strongly, or only slightly affecting CoV replication in ball format, colored red and blue, respectively. Active site residues are depicted in orange. CsA binding to the PPIase active pocket of CypA is shown in green. The calcineurin-binding surface of CypA/CsA complex is indicated schematically.

Figure 2

Replication analysis of HCoV-229E-LUC in Huh-7.5-KD and single SNP variant mutants. (a) qPCR analysis of CypA expression in sh-CypA-KD, non-target control (sh-Ctr) and with wtCypA reconstituted sh-CypA-KD cells, as well as sh-CypA-KD cells reconstituted with CypA SNP variants carrying amino acid exchanges at D66E, N106I, G96D, E134K, E84D and I89T. Huh-7.5 cells and subclones were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (both Gibco LifeTechnologies), l-glutamine, non-essential amino acids, penicillin, and streptomycin. Cells harboring small hairpin RNA (shRNA) constructs were kept in the presence of blasticidin (5 μg/mL). G418 (750 μg/mL) was additionally added to cells carrying pWPI-encoded CypA variants [46]. hTOP1 was used in qPCR to standardize cyclophilin expression. (b) Replication was measured by determining Renilla Luciferase activity in cell extracts after 24 and 48 h p.I. Values are given as relative light units (RLU). (c) HCoV-229E-LUC N-protein expression as replication measure in Huh-7.5, sh-CypA-KD, non-target control (sh-Ctr) and with wtCypA reconstituted Huh-7.5 cells. qPCR/Western blot methods and materials used are the same as described in [36].