Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants

In studying the subdominant status of two cysteine-containing influenza vir us nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2K(d), we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the af finity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We f ound similar effects for two widely studied, cysteine-containing peptides f rom lymphocytic choriomeningitis virus. The major modifications of cysteine -containing synthetic peptides are cysteinylation and dimerization occurrin g through cysteine residues. We demonstrate that both of these modification s occur in cells synthesizing a cytosolic NP218-226 minigene product and, f urther, that T cells specific for cysteinylated NP218-226 are induced by in fluenza virus infection in mice, demonstrating that this modification occur s in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral pep tides and that this must be considered in studying the status of such pepti des in immunodominance hierarchies.