West nile virus ligands from alternative reading frame proteins are revealed by class I HLA

Abstract

Aim Class I human leukocyte antigens alert the cellular immune system of infections by presenting peptides derived from viral proteins on the surface of infected cells. Viruses are capable of encoding alternative reading frame (ARF) proteins, and such proteins have only been partially characterized for existence and function. West Nile virus (WNV) encodes an ARF protein (NS1’) and possibly other ARF proteins. Here we use high throughput ligand analysis to characterize the class I HLA of WNV infected cells in order to identify peptides that originate from WNV ARF proteins. Methods Epithelial cells expressing different soluble HLA-A and HLA-B class I molecules were cultured in bioreactors and infected with WNV. HLA/peptide complexes were harvested from WNV infected cells, peptides were eluted from affinity-purified HLA, and two-dimensional LC/MS produced spectra were searched against a WNV ARF database using PEAKS proteomics software. The sequence identity of each WNV ARF peptide was confirmed by comparing the MS2 fragmentation of the eluted experimental ARF peptide with a corresponding synthetic peptide. Results We were able to identify and confirm 4 NS1’ derived peptides presented by HLA-A*01:01 and HLA-A∗24:02. Interestingly we were also able to discover a peptide, from a novel ARF WNV protein presented by HLA-B*35:01. This protein is likely a 21 amino acid long C-terminal extension of NS5 and is presumed to be the result of a +1 frame shift, 38 base pair upstream from the polyprotein stop codon. This viral protein/peptide is of unknown function. Conclusions We demonstrate that different HLA class I molecules present peptides from the NS1’ protein that is created as a result of ribosomal frameshifting during viral replication. In addition, we show the first evidence of presence of another ARF protein that is produced after WNV infection. How these ligands impact the immune responses toward infected cells remains to be resolved.