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Stress protein (hsp73)-mediated, TAP-independent processing of endogenous, truncated SV40 large T antigen for
R Schirmbeck1, W Böhm, J Reimann
1Institute for Medical Microbiology, University of Ulm, Germany.
Abstract:
Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the constitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two Db-binding peptides and one Kb-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two Db-binding epitopes, but not the Kb-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi Db molecules is available for 2-3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.
Insights
SV40 large tumor antigen (T-Ag) processing was investigated in TAP-competent and deficient cells. A truncated T-Ag variant associated with heat shock protein 73 (hsp73) enabled peptide presentation in TAP-deficient cells, suggesting a novel antigen processing pathway.
Area of Science:
- Immunology
- Molecular Biology
- Virology
Background:
- The transporter associated with antigen processing (TAP) is crucial for presenting viral antigens to cytotoxic T lymphocytes (CTLs).
- SV40 large tumor antigen (T-Ag) is a viral protein involved in cell transformation and immune evasion.
- Heat shock proteins (hsp73) act as cellular chaperones, influencing protein stability and processing.
Purpose of the Study:
- To investigate the role of TAP in presenting SV40 T-Ag-derived peptides.
- To determine if a truncated T-Ag variant (cT-Ag) associated with hsp73 can bypass TAP-dependent antigen presentation.
- To explore the mechanism of antigen loading onto MHC class I molecules in TAP-deficient cells.
Main Methods:
- Transfection of TAP-competent and TAP-deficient cell lines with wild-type (wt) T-Ag or truncated cT-Ag expression plasmids.
- Analysis of T-Ag expression and association with hsp73.
- Presentation of T-Ag-derived peptides to cytotoxic T lymphocyte lines (CTLLs) using flow cytometry.
Main Results:
- Both wtT-Ag and cT-Ag were stably expressed. The cT-Ag variant, but not wtT-Ag, associated with hsp73.
- TAP-competent cells presented T-Ag epitopes regardless of T-Ag form. TAP-deficient cells expressing wtT-Ag failed to present epitopes.
- TAP-deficient cells expressing hsp73-associated cT-Ag presented specific Db-binding epitopes, but not a Kb-binding epitope.
Conclusions:
- The hsp73-associated cT-Ag can be processed and presented in a TAP-independent manner, suggesting an alternative antigen processing pathway.
- A pool of post-Golgi Db molecules is available for peptide loading in TAP-deficient cells, facilitating presentation of endolysosomally processed antigens.
- This finding has implications for understanding viral immune evasion strategies and developing T-cell based immunotherapies.