Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation

Yoshinori Ito, Eisei Kondo, Ayako Demachi-Okamura, Yoshiki Akatsuka, Kunio Tsujimura, Mitsune Tanimoto, Yasuo Morishima, Toshitada Takahashi, Kiyotaka Kuzushima

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A 222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.

Original languageEnglish
Pages (from-to)883-890
Number of pages8
JournalJournal of Virology
Volume80
Issue number2
DOIs
Publication statusPublished - 01-01-2006

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Human herpesvirus 4
cytotoxic T-lymphocytes
T-Lymphocyte Epitopes
Cytotoxic T-Lymphocytes
Human Herpesvirus 4
membrane proteins
epitopes
Epitopes
Membrane Proteins
puromycin
Defective Viruses
Enzyme-Linked Immunospot Assay
Puromycin
Intrinsic Factor
Proteins
HLA-A Antigens
proteins
proteasome endopeptidase complex
amino acid substitution
Gene Silencing

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Ito, Yoshinori ; Kondo, Eisei ; Demachi-Okamura, Ayako ; Akatsuka, Yoshiki ; Tsujimura, Kunio ; Tanimoto, Mitsune ; Morishima, Yasuo ; Takahashi, Toshitada ; Kuzushima, Kiyotaka. / Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation. In: Journal of Virology. 2006 ; Vol. 80, No. 2. pp. 883-890.
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Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation. / Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka.

In: Journal of Virology, Vol. 80, No. 2, 01.01.2006, p. 883-890.

Research output: Contribution to journalArticle

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T1 - Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation

AU - Ito, Yoshinori

AU - Kondo, Eisei

AU - Demachi-Okamura, Ayako

AU - Akatsuka, Yoshiki

AU - Tsujimura, Kunio

AU - Tanimoto, Mitsune

AU - Morishima, Yasuo

AU - Takahashi, Toshitada

AU - Kuzushima, Kiyotaka

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AB - The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A 222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.

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