C-terminal fragments of cystatin C inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

Abstract

GPR15 is a GPCR reported to regulate T cell trafficking to the colon and epithelia that might play a role in intestinal homeostasis and inflammation. Thus, GPR15 is a promising target for altering T cell migration. Accumulating evidence suggests an important role of this receptor in certain types of cancer. To discover novel endogenous GPR15 ligands, I took advantage of the fact that GPR15 also serves as an entry cofactor for HIV and SIV. To this end, a library of hemofiltrate-derived peptides was analyzed for inhibitors of GPR15- mediated SIV infection, leading to the purification and identification of a C-terminal fragment of Cystatin C (CysC95-146) binding to the N-terminal regions of GPR15. CysC95- 146 and other C-terminal Cystatin C fragments are detectable in blood-derived human hemofiltrate. They are generated by digestion of the abundant plasma resident Cystatin C by proteases that are present and activated at sites of infection and inflammation. CysC95-146 inhibited GPR15-dependent entry of various HIV-1, HIV-2 and SIV strains in several cell lines and in primary CD4+ T cells. In contrast, GPR15L, the recently discovered chemokine ligand of GPR15, displayed little if any inhibitory effect. This finding was unexpected since the chemokine ligands of CCR5 and CXCR4 inhibit R5- or X4-tropic HIV-1 infection respectively. In addition, binding of CysC95-146 does not induce GPR15 signaling nor does it interfere with signal transduction by GPR15L. Thus, my results suggest that the main binding site of CysC95-146 in the N-terminus of GPR15 is crucial for HIV and SIV infection and distinct from the interaction site of GPR15L. CysC95-146 is conserved in the simian hosts of SIV infection and the macaque-derived peptide is as active as the human orthologue in inhibiting GPR15-dependent virus entry. In summary, I discovered CysC95-146 as a novel physiological ligand of GPR15. To our knowledge, it is the first known peptidic inhibitor of GPCR-mediated infection by lentiviral pathogens that does not interfere with the signaling function of the corresponding chemokine receptor. It is derived from Cystatin C, a highly abundant cysteine protease inhibitor, and might be generated locally in vivo in response to infection and inflammation. In addition, CysC95-146 fulfills the requirements of a diagnostic agent with high specificity for GPR15. Since this peptide does not compromise the physiological function of GPR15, it might be applicable to monitor GPR15 expression e.g. in tumor tissue or to deliver bioactive molecules to GPR15 expressing cells without altering or blocking the receptor in the process.