Mooring Stone-Like Arg Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 10101 (SLA-10101). We found that peptide binding in SLA-10101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-10101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-10101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-10101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-10101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 10101 (SLA-10101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-10101 revealed an unusual peptide anchoring mode of SLA-10101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-10101-binding peptides. Notably, we determined an unexpected role of Arg of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.